Nucleic acids encoding human antibodies to sialyl-lewis a

ABSTRACT

The present invention provides compositions for the production of an antibody or functional fragment thereof directed against Sialyl-Lewis a  (sLe a ). The compositions of the invention include polynucleotides encoding a heavy chain and/or a light chain variable domain that binds to sLe a . The invention also provides an isolated antibody or functional fragment thereof and methods of treating or preventing a disease, such as cancer or tumor formation, wherein the antibody or functional fragment includes a variable heavy chain domain and a variable light chain domain that has an amino acid sequence provided herein. The invention further provides a conjugate of an antibody or functional fragment thereof conjugated or recombinantly fused to a diagnostic agent, detectable agent or therapeutic agent, and methods of treating, preventing or diagnosing a disease in a subject in need thereof.

This application is a continuation of application Ser. No. 16/788,251, filed Feb. 11, 2020, which is a continuation of application Ser. No. 15/275,174, filed Sep. 23, 2016, which is a continuation of application Ser. No. 14/468,827, filed Aug. 26, 2014, which claims the benefit of priority of U.S. Provisional Application Ser. No. 61/870,137, filed Aug. 26, 2013, the entire contents of which is incorporated herein by reference.

This invention was made with government support under grant number CA-128362 awarded by the National Cancer Institute, NIH. The government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted in XML format via EFS-Web and is hereby incorporated by reference in its entirety. Said XML copy, created on Jul. 14, 2023, is named 2013237-0571_SL.xml and is 32,414 bytes in size.

BACKGROUND OF THE INVENTION

The present invention relates generally to antibodies directed against Sialyl-Lewis^(a) (sLe^(a)), and more specifically to polynucleotides encoding anti-sLe^(a) antibodies and the corresponding encoded antibodies or fragments thereof.

Passive administration of antibodies directed against tumor specific antigens may eliminate tumor cells and early metastases during cancer development. This treatment may also have a significant impact on cancer recurrence. Antibodies directed against tumor specific carbohydrates may be useful candidates in this cancer treatment. For example, many tumor-restricted monoclonal antibodies resulting from immunization of mice with human cancer cells have been shown to be directed against carbohydrate antigens expressed at the cell surface as glycolipids or glycoproteins. The carbohydrate sLe^(a) has been shown to be expressed on tumors of the gastrointestinal tract. Expression of sLe^(a) has also been shown to impact metastatic potential and correlates with increased metastatic potential in human colon cancer and pancreatic adenocarcinoma. However, carbohydrate chemistry has been rather challenging and the clinical development of antibodies that recognize such tumor specific carbohydrates has been slow.

Pancreatic carcinoma is one of the most aggressive adenocarcinomas and is often associated with a poor prognosis. Pancreatic carcinoma ranks as the fourth leading cause of cancer mortality. Despite advances in the screening for different carcinomas, the reliability of detecting malignant lesions stemming from the pancreas remains poor. Positron emission tomography utilizing fluorodeoxyglucase (FDG-PET) has been indicated for the detection and staging of pancreatic cancer. However, FDG-PET is insensitive to differentiating pancreatitis from malignancy and remains problematic in staging small primary lesions (<7 mm) and liver metastases (<1 cm). One diagnostic screening method used to monitor the state of pancreatic ductal adenocarcinoma (PDAC) patients includes detecting elevated levels of circulating sLe^(a) antigen in sera. Patients with >37 U/ml of circulating sLe^(a) antigen indicates cancer recurrence. However, development of alternative diagnostic tools that utilize such tumor specific carbohydrates has been slow.

Thus, there exists a need for identifying and generating antibodies that specifically recognize tumor specific carbohydrates, such as sLe^(a), for the treatment of recurring cancers and for detecting malignant lesions and metastases. This invention satisfies this need and provides related advantages.

SUMMARY OF INVENTION

In accordance with the present invention, herein provided are compositions for producing antibodies or functional fragments thereof that bind sLe^(a). The compositions include an isolated polynucleotide encoding an antibody or a functional fragment thereof, wherein the antibody includes a variable heavy chain (VH) domain that has an amino acid sequence provided herein. The isolated polynucleotide of the invention can also include a nucleic acid sequence provided herein, wherein the nucleic acid sequence encodes the VH domain of the antibody or functional fragment thereof.

In another embodiment of the invention, the isolated polynucleotide can encode an antibody or a functional fragment thereof, wherein the antibody includes a variable light chain (VL) domain that has an amino acid sequence provided herein. The isolated polynucleotide of the invention can also include a nucleic acid sequence provided herein, wherein the nucleic acid sequence encodes the VL domain of the antibody or functional fragment thereof.

The compositions of the invention also include an isolated antibody or functional fragment thereof, wherein the antibody binds to sLe^(a). In some embodiments, the invention provides an isolated antibody or functional fragment thereof that binds to sLe^(a), wherein the antibody or functional fragment thereof includes a VH domain having an amino acid sequence provided herein.

In some embodiments, the invention provides an isolated antibody or functional fragment thereof that binds to sLe^(a), wherein the antibody or functional fragment thereof includes a VL domain having an amino acid sequence provided herein.

In some embodiments, the invention provides an isolated antibody or functional fragment thereof that binds to sLe^(a), wherein the antibody or functional fragment thereof includes both a VH domain and a VL domain, where the VH domain and the VL domain respectively include an amino acid sequence for the respective VH and VL domains of the clonal isolates provided herein.

In some embodiments, the invention provides a conjugate having an antibody or functional fragment provided herein that is conjugated or recombinantly fused to a diagnostic agent, detectable agent or therapeutic agent. In some aspects of the invention, a conjugate of the invention that includes a detectable agent can be used in a method for detecting and/or diagnosing tumor formation is a subject. Such methods can include administering an effective amount of the conjugate to a subject in need thereof.

In some embodiments, the invention provides pharmaceutical compositions having one or more antibody or functional fragment of the invention and a pharmaceutically acceptable carrier. In some aspects, the invention also provides a method for treating or preventing a disease in a subject in need thereof, by administering a therapeutically effective amount of a pharmaceutical composition of the invention. In still another aspect, the invention provides administering a second therapeutic agent concurrently or successively with an antibody or functional fragment of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequence and the encoded amino acid sequence of the variable heavy (VH) chain domain of clone 5B1 and a leader sequence that can be used for recombinant expression. The top portion of the figure shows an alignment between the nucleotide sequence of SEQ ID NO: 1 and amino acid sequence of SEQ ID NO: 2. The three complementarity determining regions (CDR1, CDR2 and CDR3) are also identified.

FIG. 2 shows the nucleotide sequence and the encoded amino acid sequence of the variable light (VL) chain domain of clone 5B1 and a leader sequence that can be used for recombinant expression. The top portion of the figure shows an alignment between the nucleotide sequence of SEQ ID NO: 3 and amino acid sequence of SEQ ID NO: 4. The three complementarity determining regions (CDR1, CDR2 and CDR3) are also identified.

FIG. 3 shows the nucleotide sequence and the encoded amino acid sequence of the variable heavy (VH) chain domain of clone 9H3 and a leader sequence that can be used for recombinant expression. The top portion of the figure shows an alignment between the nucleotide sequence of SEQ ID NO: 5 and amino acid sequence of SEQ ID NO: 6. The three complementarity determining regions (CDR1, CDR2 and CDR3) are also identified.

FIG. 4 shows the nucleotide sequence and the encoded amino acid sequence of the variable light (VL) chain domain of clone 9H3 and a leader sequence that can be used for recombinant expression. The top portion of the figure shows an alignment between the nucleotide sequence of SEQ ID NO: 7 and amino acid sequence of SEQ ID NO: 8. The three complementarity determining regions (CDR1, CDR2 and CDR3) are also identified.

FIG. 5 shows the nucleotide sequence and the encoded amino acid sequence of the variable heavy (VH) chain domain of clone 5H11 and a leader sequence that can be used for recombinant expression. The top portion of the figure shows an alignment between the nucleotide sequence of SEQ ID NO: 9 and amino acid sequence of SEQ ID NO: 10. The three complementarity determining regions (CDR1, CDR2 and CDR3) are also identified.

FIG. 6 shows the nucleotide sequence and the encoded amino acid sequence of the variable light (VL) chain domain of clone 5H11 and a leader sequence that can be used for recombinant expression. The top portion of the figure shows an alignment between the nucleotide sequence of SEQ ID NO: 11 and amino acid sequence of SEQ ID NO: 12. The three complementarity determining regions (CDR1, CDR2 and CDR3) are also identified.

FIG. 7 shows the nucleotide sequence and the encoded amino acid sequence of the variable heavy (VH) chain domain of clone 7E3 and a leader sequence that can be used for recombinant expression. The top portion of the figure shows an alignment between the nucleotide sequence of SEQ ID NO: 13 and amino acid sequence of SEQ ID NO: 14. The three complementarity determining regions (CDR1, CDR2 and CDR3) are also identified.

FIG. 8 shows the nucleotide sequence and the encoded amino acid sequence of the variable light (VL) chain domain of clone 7E3 and a leader sequence that can be used for recombinant expression. The top portion of the figure shows an alignment between the nucleotide sequence of SEQ ID NO: 15 and amino acid sequence of SEQ ID NO: 16. The three complementarity determining regions (CDR1, CDR2 and CDR3) are also identified.

FIG. 9 shows the nucleotide sequence and the encoded amino acid sequence of a diabody designated 5B1CysDb having CDR1, CDR2 and CDR2 of both the variable heavy (VH) and variable light (VL) chain domains of clone 5B1. The top portion of the figure shows an alignment between the nucleotide sequence of SEQ ID NO: 17 and amino acid sequence of SEQ ID NO: 18. The three complementarity determining regions (CDR1, CDR2 and CDR3) for both the VH and VL domains are identified in bold and underline text. The linker sequence and polyhistidine tag (Poly His-Tag) with added amino acids are also indicated by italic and underline text.

FIG. 10 shows the nucleotide sequence and the encoded amino acid sequence of a diabody designated 7E3CysDb having CDR1, CDR2 and CDR2 of both the variable heavy (VH) and variable light (VL) chain domains of clone 7E3. The top portion of the figure shows an alignment between the nucleotide sequence of SEQ ID NO: 19 and amino acid sequence of SEQ ID NO: 20. The three complementarity determining regions (CDR1, CDR2 and CDR3) for both the VH and VL domains are identified in bold and underline text. The linker sequence and polyhistidine tag (Poly His-Tag) with added amino acids are also indicated by italic and underline text.

FIGS. 11A-11F show the binding of human anti-sLe^(a) antibodies to tumor cells analyzed by flow cytometry. FIG. 11A shows DMS-79 cells stained with recombinant (r) 5B1, 9H3, 5H11, and 7E3 antibodies. FIGS. 11B-11F respectively shows HT29, BxPC3, SW626, SK-MEL28, and Colo205-luc cells stained with 1-2 μg/mL of r5B1 or r7E3 plus IgG or IgM-specific secondary antibody as described in Example I.

FIGS. 12A and 12B show CDC activity of r5B1 and r7E3 antibodies in comparison to murine 121SLE (IgM) in the presence of human complement (Hu C′) as measured against DMS-79 cells. Human isotype control antibodies, Hu IgG (⋄) and Hu IgM (♦) showed <4% cytotoxicity. A dose response for r5B1 IgG (▪), r7E3 IgM (●) and 121SLE mIgM (▴) antibodies is shown in FIG. 12A. The calculated EC50 (μg/ml) for r5B1 (IgG), r7E3 (IgM) and 121SLE (mIgM) antibodies is shown in FIG. 12B.

FIGS. 13A-13C show antibody-dependent cell-mediated cytotoxicity (ADCC) of r5B1 antibodies. FIG. 13A shows r5B1-mediated ADCC with human PBMC against DMS-79 cells. PBMC were tested at E:T ratios from 100:1 to 12.5:1 with DMS-79 tumor cells in the presence or absence of 2 μg/mL r5B1. FIG. 13B shows r5B1-mediated ADCC with primary human NK cells against DMS-79 cells. NK cells were tested at lower E:T ratios from 5:1 to 0.6:1 with DMS-79 tumor cells in the presence or absence of 2 μg/mL r5B1. FIG. 13C shows ADCC of r5B1 at various concentrations with PBMCs from 2 donors at an E:T ratio of 1:100 with DMS-79 tumor cells in the presence of the indicated concentrations of r5B1.

FIG. 14 shows internalization of sLe^(a) into BxPC3 cells. BxPC3 pancreatic tumor cells were grown in the presence of r5B1 (anti-sLe^(a)) or r1B7 (anti-GD2) antibodies complexed with Hum-ZAP, a saporin-conjugated anti-human IgG. After 3 days, the viability of the cells was measured using an 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the sample values were normalized to the values of untreated cultures.

FIG. 15 shows activity of r5B1 antibody in a xenograft model using Colo205-luc cells. Severe combined immunodeficient (SCID) mice (5 per group) received 0.5 million Colo205-luc cells by tail vein injection on day 0. Mice received 100 μg r5B1 by intraperitoneal injection on days 1, 7, 14, and 21 (experiment 1, Exp1) or on days 1, 4, 7, 10, 14, and 21 (experiment 2, Exp2) for a total dose of 600 μg. Control (Ctrl) animals received PBS mock injections.

FIG. 16 shows the effect of r5B1 on Colo205-luc tumors in SCID mice. Mice received 100 μg (▾), 300 μg (▪) or 1 mg (♦) r5B1 antibody per injection as described in Example I. Control (▪) animals received PBS mock injections.

FIG. 17 shows the fluorescence imaging of five mice per group for r5B1 treated mice having Colo205-luc tumors at Day 0 and Week 5. The mice received the treatment regiment depicted in FIG. 16 and described in Example I.

FIGS. 18A and 18B show the anti-tumor activity in a therapeutic subcutaneous xenograft model using DMS-79 cells. FIG. 18A shows the suppression or regression of 5B1 treated mice (5B1 alone (▴) or 5B1+cRGD (▾)) in comparison to Human IgG (IgG alone (♦) or IgG+cRGD (◯)) and PBS injected control (▪). Arrows indicate days of antibody or PBS injections. FIG. 18B shows representative images of treated mice. Arrows indicate absence of any visual tumor.

FIGS. 19A-19F show binding of 5B1 to various tumor types. FIG. 19A is a pancreas, ductal adenocarcinoma, stage III tumor. FIG. 19B is a sigmoid colon, carcinoma stage TIM tumor. FIG. 19C is a lung, adenocarcinoma, stage D3 tumor. FIG. 19D is a urinary bladder, mucinous adenocarcinoma, stage IV tumor. FIG. 19E is a ovary, metastatic carcinoma from colon tumor. FIG. 19F is a lymph node, metastatic carcinoma, stage IIIA tumor.

FIG. 20 shows serial PET maximum intensity projection (MIP) images acquired from 2-120 h with ⁸⁹Zr radiolabed-5B1 antibody (⁸⁹Zr-5B1) intravenously administered to female SCID mice subcutaneously implanted with BxPC3 pancreatic tumors. PET-MIP imaging demonstrates high tumor uptake with clearance of non-specifically bound tracer as early as 24 hours post injection (h p.i.)

FIG. 21 shows biodistribution results that are in agreement with the PET data of FIG. 20 , with an observed tumor uptake of 84.73±12.28% ID/g. Because of the small tumor weights, a plot of tumor uptake expressed as % ID versus time is displayed by the inset graph. The tumor % ID display significant tumor uptake by ⁸⁹Zr-5B1 at all time points, and, is at least seven-fold greater than non-specific ⁸⁹Zr-IgG. Competitive inhibition with cold 5B1 (200 μg) show a decrease in tumor accumulation.

FIGS. 22A-22C show PET-MIP images of mice-bearing DMS79 (FIG. 22A) and Colo205-luc xenografts (FIG. 22B). PET-MIP imaging delineation of tumor (T), heart (H) and liver (L) by ⁸⁹Zr-5B1 are indicated. The colorectal Colo205-luc xenografts model displays ⁸⁹Zr-5B1 accumulation peaking at 24 h, which eventually decreases while an increase in non-specific binding to the liver was exhibited (FIG. 22C).

FIG. 23 shows a dose dependent inhibition and regression of tumor growth in a DMS-79 small lung cell carcinoma xenograft model treated with successive co-administration of 5B1 antibody and Taxol (Paclitaxel). Large arrows on the X axis indicate 5B1 treatment. Co-administration of 5B1 antibody and Taxol significantly limited tumor growth and resulted in tumor regression in comparison to control human IgG (HuIgG) or 5B1 antibody and Taxol administered individually. Significantly differences from control by 2-way ANOVA at p<0.01 (**) and p<0.001 (***) are indicated. N=5.

FIG. 24 shows the inhibition of tumor growth in a BxPc3 pancreatic carcinoma xenograft model treated with successive co-administration of 5B1 antibody and Taxol (Paclitaxel). Large arrows on the X axis indicate Taxol plus 5B1 treatment, whereas the small arrows indicate 5B1 alone treatment. Co-administration of 5B1 antibody and Taxol significantly limited tumor growth in comparison to controls (PBS-Ctrl; human IgG-HuIgG) or 5B1 antibody and Taxol administered individually.

FIGS. 25A and 25B, show representative images of mice that were orthotopically transplanted with BxPC3-luc pancreatic tumor xenografts. FIG. 25A: The co-registration of FDG-PET and computed tomography (CT) (left) and planar sections of FDG-PET only (right) displayed minimal tumor detection of the tracer with a high uptake in highly metabolic tissues (i.e. heart, H and bladder, B). FIG. 25B: Acquired ⁸⁹Zr radiolabed-5B1 antibody (⁸⁹Zr-5B1) PET image of the same mouse co-registered with CT exhibited exceptional tumor detection of the BxPC3-luc tumor xenografts.

DETAILED DESCRIPTION OF THE INVENTION

Carbohydrates expressed on the tumor cell surface can be targets for passive immunotherapy. The compositions provided herein are based, at least in part, on the identification and characterization of human antibodies that were generated from blood lymphocytes of individuals immunized with a Sialyl-Lewis^(a)-keyhole limpet hemocyanin (sLe^(a)-KLH) conjugate vaccine. At least four antibodies with high affinity for sLe^(a) (5B1, 9H3, 5H11 and 7E3) were identified. Two of these antibodies were expressed as recombinant antibodies (r5B1 and r7E3) and further characterized in in vitro and in vivo models. Both antibodies were potent in complement-dependent cytotoxicity (CDC) assays, and the 5B1 antibody was also highly active in antibody-dependent cytotoxicity assays. The in vivo efficacy of the antibodies were tested in two xenograft models using either Colo205 tumor cells or DMS-79 tumor cells engrafted into severe combined immunodeficient (SCID) mice. The translational relevance of the invention provided herein is 2 fold: First, the approach provided herein demonstrates that the antibody response elicited by a sLe^(a)-KLH vaccine is useful as a vaccine itself. Second, the most potent antibodies that were generated in a clinical trial can be preserved and ultimately used as therapeutics, or in the generation of therapeutics, for a target cancer population. The high affinity of the antibodies provided herein and their high effector functions support this translational potential.

As used herein, the term “antibody” is intended to mean a polypeptide product of B cells within the immunoglobulin class of polypeptides that is able to bind to a specific molecular antigen and is composed of two identical pairs of polypeptide chains, wherein each pair has one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa) and each amino-terminal portion of each chain includes a variable region of about 100 to about 130 or more amino acids and each carboxy-terminal portion of each chain includes a constant region (See Borrebaeck (ed.) (1995) Antibody Engineering, Second Edition, Oxford University Press.; Kuby (1997) Immunology, Third Edition, W.H. Freeman and Company, New York). In the context of the present invention, the specific molecular antigen that can be bound by an antibody of the invention includes the target carbohydrate sLe^(a).

The term “human” when used in reference to an antibody or a functional fragment thereof refers an antibody or functional fragment thereof that has a human variable region and/or a human constant region or a portion thereof corresponding to human germline immunoglobulin sequences. Such human germline immunoglobulin sequences are described by Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242. A human antibody, in the context of the present invention, can include an antibody that binds to sLe^(a) and is encoded by a nucleic acid sequence that is a naturally occurring somatic variant of the human germline immunoglobulin nucleic acid sequence. Exemplary methods of producing human antibodies are provided in Example I, but any method well known to those skilled in the art can be used.

The term “monoclonal antibody” refers to an antibody that is the product of a single cell clone or hybridoma or a population of cells derived from a single cell. A monoclonal antibody also is intended to refer to an antibody produced by recombinant methods from heavy and light chain encoding immunoglobulin genes to produce a single molecular immunoglobulin species. Amino acid sequences for antibodies within a monoclonal antibody preparation are substantially homogeneous and the binding activity of antibodies within such a preparation exhibit substantially the same antigen binding activity. In contrast, polyclonal antibodies are obtained from different B cells within a population, which are a combination of immunoglobulin molecules that bind a specific antigen. Each immunoglobulin of the polyclonal antibodies can bind a different epitope of the same antigen. Methods for producing both monoclonal antibodies and polyclonal antibodies are well known in the art (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989) and Borrebaeck (ed.), Antibody Engineering: A Practical Guide, W.H. Freeman and Co., Publishers, New York, pp. 103-120 (1991)).

As used herein, the term “functional fragment” when used in reference to an antibody is intended to refer to a portion of the antibody including heavy or light chain polypeptides that retains some or all of the binding activity as the antibody from which the fragment was derived. Such functional fragments can include, for example, an Fd, Fv, Fab, F(ab′), F(ab)₂, F(ab′)₂, single chain Fv (scFv), diabody, triabody, tetrabody and minibody. Other functional fragments can include, for example, heavy or light chain polypeptides, variable region polypeptides or CDR polypeptides or portions thereof so long as such functional fragments retain binding activity. Such antibody binding fragments can be found described in, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989); Myers (ed.), Molec. Biology and Biotechnology: A Comprehensive Desk Reference, New York: VCH Publisher, Inc.; Huston et al., Cell Biophysics, 22:189-224 (1993); Plückthun and Skerra, Meth. Enzymol., 178:497-515 (1989) and in Day, E. D., Advanced Immunochemistry, Second Ed., Wiley-Liss, Inc., New York, NY (1990).

The term “heavy chain” when used in reference to an antibody refers to a polypeptide chain of about 50-70 kDa, wherein the amino-terminal portion includes a variable region of about 120 to 130 or more amino acids and a carboxy-terminal portion that includes a constant region. The constant region can be one of five distinct types, referred to as alpha (α), delta (δ), epsilon (ε), gamma (γ) and mu (μ), based on the amino acid sequence of the heavy chain constant region. The distinct heavy chains differ in size: α, δ and γ contain approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. When combined with a light chain, these distinct types of heavy chains give rise to five well known classes of antibodies, IgA, IgD, IgE, IgG and IgM, respectively, including four subclasses of IgG, namely IgG1, IgG2, IgG3 and IgG4. A heavy chain can be a human heavy chain.

The term “light chain” when used in reference to an antibody refers to a polypeptide chain of about 25 kDa, wherein the amino-terminal portion includes a variable region of about 100 to about 110 or more amino acids and a carboxy-terminal portion that includes a constant region. The approximate length of a light chain is 211 to 217 amino acids. There are two distinct types, referred to as kappa (κ) of lambda (λ) based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art. A light chain can be a human light chain.

The term “variable domain” or “variable region” refers to a portion of the light or heavy chains of an antibody that is generally located at the amino-terminal of the light or heavy chain and has a length of about 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids in the light chain, and are used in the binding and specificity of each particular antibody for its particular antigen. The variable domains differ extensively in sequence between different antibodies. The variability in sequence is concentrated in the CDRs while the less variable portions in the variable domain are referred to as framework regions (FR). The CDRs of the light and heavy chains are primarily responsible for the interaction of the antibody with antigen. Numbering of amino acid positions used herein is according to the EU Index, as in Kabat et al. (1991) Sequences of proteins of immunological interest. (U.S. Department of Health and Human Services, Washington, D.C.) 5^(th) ed. A variable region can be a human variable region.

A CDR refers to one of three hypervariable regions (H1, H2 or H3) within the non-framework region of the immunoglobulin (Ig or antibody) VH β-sheet framework, or one of three hypervariable regions (L1, L2 or L3) within the non-framework region of the antibody VL β-sheet framework. Accordingly, CDRs are variable region sequences interspersed within the framework region sequences. CDR regions are well known to those skilled in the art and have been defined by, for example, Kabat as the regions of most hypervariability within the antibody variable (V) domains (Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat, Adv. Prot. Chem. 32:1-75 (1978)). CDR region sequences also have been defined structurally by Chothia as those residues that are not part of the conserved β-sheet framework, and thus are able to adapt different conformations (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). Both terminologies are well recognized in the art. The positions of CDRs within a canonical antibody variable domain have been determined by comparison of numerous structures (Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); Morea et al., Methods 20:267-279 (2000)). Because the number of residues within a hypervariable region varies in different antibodies, additional residues relative to the canonical positions are conventionally numbered with a, b, c and so forth next to the residue number in the canonical variable domain numbering scheme (Al-Lazikani et al., supra (1997)). Such nomenclature is similarly well known to those skilled in the art.

For example, CDRs defined according to either the Kabat (hypervariable) or Chothia (structural) designations, are set forth in the Table 1 below.

TABLE 1 CDR Definitions Kabat¹ Chothia² Loop Location V_(H) CDR1 31-35 26-32 linking B and C strands V_(H) CDR2 50-65 53-55 linking C′ and C″ strands V_(H) CDR3  95-102  96-101 linking F and G strands V_(L) CDR1 24-34 26-32 linking B and C strands V_(L) CDR2 50-56 50-52 linking C′ and C″ strands V_(L) CDR3 89-97 91-96 linking F and G strands ¹Residue numbering follows the nomenclature of Kabat et al., supra ²Residue numbering follows the nomenclature of Chothia et al., supra

One or more CDRs also can be incorporated into a molecule either covalently or noncovalently to make it an immunoadhesin. An immunoadhesin can incorporate the CDR(s) as part of a larger polypeptide chain, can covalently link the CDR(s) to another polypeptide chain, or can incorporate the CDR(s) noncovalently. The CDRs permit the immunoadhesin to bind to a particular antigen of interest.

As used herein, the term “isolated” when used in reference to an antibody, antibody functional fragment or polynucleotide is intended to mean that the referenced molecule is free of at least one component as it is found in nature. The term includes an antibody, antibody functional fragment or polynucleotide that is removed from some or all other components as it is found in its natural environment. Components of an antibody's natural environment include, for example, erythrocytes, leukocytes, thrombocytes, plasma, proteins, nucleic acids, salts and nutrients. Components of an antibody functional fragment's or polynucleotide's natural environment include, for example, lipid membranes, cell organelles, proteins, nucleic acids, salts and nutrients. An antibody, antibody functional fragment or polynucleotide of the invention can also be free or all the way to substantially free from all of these components or any other component of the cells from which it is isolated or recombinantly produced.

As used herein, “isotype” refers to the antibody class that is encoded by heavy chain constant region genes. The heavy chains of a given antibody or functional fragment determine the class of that antibody or functional fragment: IgM, IgG, IgA, IgD or IgE. Each class can have either κ or λ light chains. The term “subclass” refers to the minor differences in amino acid sequences of the heavy chains that differentiate the subclasses. In humans there are two subclasses of IgA (subclasses IgA1 and IgA2) and there are four subclasses of IgG (subclasses IgG1, IgG2, IgG3 and IgG4). Such classes and subclasses are well known to those skilled in art.

The terms “binds” or “binding” as used herein refer to an interaction between molecules to form a complex. Interactions can be, for example, non-covalent interactions including hydrogen bonds, ionic bonds, hydrophobic interactions, and/or van der Waals interactions. A complex can also include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions or forces. Binding of an antibody or functional fragment thereof can be detected using, for example, an enzyme-linked immunosorbant assay, a method provided in Example I or any one of a number of methods that are well known to those skilled in the art.

The strength of the total non-covalent interactions between a single antigen-binding site on an antibody or functional fragment and a single epitope of a target molecule, such as sLe^(a), is the affinity of the antibody or functional fragment for that epitope. The ratio of association (k₁) to dissociation (k⁻¹) of an antibody or functional fragment thereof to a monovalent antigen (k₁/k⁻¹) is the association constant K, which is a measure of affinity. The value of K varies for different complexes of antibody or functional fragment and antigen and depends on both k₁ and k⁻¹. The association constant K for an antibody or functional fragment of the invention can be determined using any method provided herein or any other method well known to those skilled in the art.

The affinity at one binding site does not always reflect the true strength of the interaction between an antibody or functional fragment and an antigen. When complex antigens containing multiple, repeating antigenic determinants, such as a polyvalent sLe^(a), come in contact with antibodies containing multiple binding sites, the interaction of antibody or functional fragment with antigen at one site will increase the probability of a reaction at a second site. The strength of such multiple interactions between a multivalent antibody and antigen is called the avidity. The avidity of an antibody or functional fragment can be a better measure of its binding capacity than is the affinity of its individual binding sites. For example, high avidity can compensate for low affinity as is sometimes found for pentameric IgM antibodies, which can have a lower affinity than IgG, but the high avidity of IgM, resulting from its multivalence, enables it to bind antigen effectively.

The specificity of an antibody or functional fragment thereof refers to the ability of an individual antibody or functional fragment thereof to react with only one antigen. An antibody or functional fragment can be considered specific when it can distinguish differences in the primary, secondary or tertiary structure of an antigen or isomeric forms of an antigen.

The term “polynucleotide” refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. The sequence of a polynucleotide is composed of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA. Thus, the terms “nucleotide sequence” or “nucleic acid sequence” is the alphabetical representation of a polynucleotide. A polynucleotide can include a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. Polynucleotide also refers to both double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of this invention that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form. It is understood that the isolated polynucleotides and nucleic acids described herein are directed to non-naturally occurring polynucleotides and nucleic acids. Non-naturally occurring polynucleotides and nucleic acids can include, but are not limited to, cDNA and chemically synthesized molecules.

The term “encode” or grammatical equivalents thereof as it is used in reference to polynucleotides refers to a polynucleotide in its native state or when manipulated by methods well known to those skilled in the art that can be transcribed to produce mRNA, which is then translated into a polypeptide and/or a fragment thereof. The antisense strand is the complement of such a polynucleotide, and the encoding sequence can be deduced therefrom.

The phrase “therapeutic agent” refers to any agent that can be used in the treatment, management or amelioration of a disease associated with expression of sLe^(a) and/or a symptom related thereto. In certain embodiments, a therapeutic agent refers to an antibody or functional fragment of the invention. In other embodiments, a therapeutic agent refers to an agent other than an antibody or functional fragment of the invention. A therapeutic agent can be an agent which is well known to be useful for, or has been or is currently being used for the treatment, management or amelioration of a disease associated with expression of sLe^(a) and/or one or more symptoms related thereto.

The phrase “diagnostic agent” refers to a substance administered to a subject that aids in the diagnosis of a disease. Such substances can be used to reveal, pinpoint, and/or define the localization of a disease causing process. In certain embodiments, a diagnostic agent includes a substance that is conjugated to an antibody or functional fragment of the invention, that when administered to a subject or contacted to a sample from a subject aids in the diagnosis of cancer or tumor formation.

The phrase “detectable agent” refers to a substance that can be used to ascertain the existence or presence of a desired molecule, such as an antibody or functional fragment of the invention, in a sample or subject. A detectable agent can be a substance that is capable of being visualized or a substance that is otherwise able to be determined and/or measured (e.g., by quantitation).

An “effective amount” is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages. Such delivery is dependent on a number of variables including the time period for which the individual dosage unit is to be used, the bioavailability of the agent, the route of administration, etc.

The phrase “therapeutically effective amount” as used herein refers to the amount of a therapeutic agent (e.g., an antibody or functional fragment provided herein or any other therapeutic agent provided herein) which is sufficient to reduce and/or ameliorate the severity and/or duration of a given disease and/or a symptom related thereto. A therapeutically effective amount of a therapeutic agent can be an amount necessary for the reduction or amelioration of the advancement or progression of a given disease, reduction or amelioration of the recurrence, development or onset of a given disease, and/or to improve or enhance the prophylactic or therapeutic effect of another therapy (e.g., a therapy other than the administration of an antibody or functional fragment provided herein).

The compound “Sialyl-Lewis^(a)” (sLe^(a)), also known as sialyl Le^(a), Sialyl-Lewis A, Sialylated Lewis a and CA 19.9, is a tetrasaccharide with a molecular formula of C₃₁H₅₂N₂O₂₃ and a molar mass of 820.74 g/mol. The structure of sLe^(a) can include Neu5Acα2-3Galβ1-3(Fucα1-4)G1cNAcβ and Neu5Gcα2-3Galβ1-3(Fucα1-4)G1cNAcβ. sLe^(a) is widely expressed on tumors of the gastrointestinal tract and is used as a tumor marker in pancreatic and colon cancer. sLe^(a) is also a known ligand for E-selection, also known as endothelial leukocyte adhesion molecule (ELAM).

In some embodiments, the present invention provides an isolated polynucleotide encoding an antibody heavy or light chain or a functional fragment thereof, wherein an antibody or functional fragment thereof generated using the antibody heavy or light chain binds to sLe^(a). Accordingly, in some embodiments, the invention provides an isolated polynucleotide encoding an antibody or a functional fragment thereof, wherein the antibody includes a VH domain that has an amino acid sequence selected from the group consisting of residues 20-142 of SEQ ID NO: 2, residues 20-142 of SEQ ID NO: 6, residues 20-142 of SEQ ID NO: 10, and residues 20-145 of SEQ ID NO: 14. The isolated polynucleotide of the invention can also include a nucleic acid sequence of residues 58-426 of SEQ ID NO: 1, residues 58-426 of SEQ ID NO: 5, residues 58-426 of SEQ ID NO: 9 or residues 58-435 of SEQ ID NO: 13, wherein the nucleic acid sequence encodes the VH domain of the antibody or functional fragment thereof.

In another embodiment of the invention, the isolated polynucleotide can encode an antibody or a functional fragment thereof, wherein the antibody includes a VL domain that has an amino acid sequence selected from the group consisting of residues 20-130 of SEQ ID NO: 4, residues 20-129 of SEQ ID NO: 8, residues 20-130 of SEQ ID NO: 12, and residues 23-130 of SEQ ID NO: 16. The isolated polynucleotide of the invention can also include a nucleic acid sequence of residues 58-390 of SEQ ID NO: 3, residues 58-387 of SEQ ID NO: 7, residues 58-390 of SEQ ID NO: 11 or residues 67-390 of SEQ ID NO: 15, wherein the nucleic acid sequence encodes the VL domain of the antibody or functional fragment thereof.

In another embodiment, the invention provides an isolated polynucleotide encoding an antibody heavy or light chain or a functional fragment thereof, wherein the antibody heavy or light chain or functional fragment thereof encoded by the polynucleotide of the invention has one or more of the complementarity determining regions (CDRs) depicted in FIGS. 1-8 or listed in Table 2. An antibody or functional fragment thereof that includes one or more of the CDRs can specifically bind to sLe^(a) as described herein. Specific binding to sLe^(a) can include the specificity, affinity and/or avidity as provided in Example I for any of the antibodies provided herein. In another aspect, an antibody or functional fragment thereof encoded by the polynucleotides of the invention can include the complement dependent cytotoxicity (CDC) activity and/or antibody-dependent cell-mediated cytotoxicity (ADCC) activity of any one of the clonal isolates 5B1, 9H3, 5H11 or 7E3 described herein. Methods for assessing the specificity, affinity and/or avidity of an antibody or functional fragment thereof are well known in the art and exemplary methods are provided herein.

TABLE 2 CDRs of Clonal Isolates Nucleic Acid Residues Amino Acid Residues Variable (SEQ ID NO:) (SEQ ID NO:) Domain CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 5B1 VH 133-156 208-231 346-393 55-62 70-77 116-131 (NO: 1) (NO: 1) (NO: 1) (NO: 2) (NO: 2) (NO: 2) 5B1 VL 133-156 208-216 325-360 45-52 70-72 109-120 (NO: 3) (NO: 3) (NO: 3) (NO: 4) (NO: 4) (NO: 4) 9H3 VH 133-156 208-231 346-393 45-52 70-77 116-131 (NO: 5) (NO: 5) (NO: 5) (NO: 6) (NO: 6) (NO: 6) 9H3 VL 133-156 208-216 325-357 45-52 70-72 109-119 (NO: 7) (NO: 7) (NO: 7) (NO: 8) (NO: 8) (NO: 8) 5H11 VH 133-156 208-231 346-393 45-52 70-77 116-131 (NO: 9) (NO: 9) (NO: 9) (NO: 10) (NO: 10) (NO: 10) 5H11 VL 134-156 208-216 325-360 45-52 70-72 109-120 (NO: 11) (NO: 11) (NO: 11) (NO: 12) (NO: 12) (NO: 12) 7E3 VH 133-156 208-231 346-402 45-52 70-77 116-134 (NO: 13) (NO: 13) (NO: 13) (NO: 13) (NO: 13) (NO: 14) 7E3 VK 145-162 214-222 331-360 49-53 72-74 111-120 (NO: 15) (NO: 15) (NO: 15) (NO: 16) (NO: 16) (NO: 16)

In some embodiments, the antibody or functional fragment thereof of the invention includes less than six CDRs. In some embodiments, the antibody or functional fragment thereof includes one, two, three, four, or five CDRs selected from the group consisting of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3. In specific embodiments, the antibody or functional fragment thereof includes one, two, three, four, or five CDRs selected from the group consisting of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 of clonal isolates 5B1, 9H3, 5H11 or 7E3 described herein.

In some embodiments, the invention provides an isolated polynucleotide that encodes an antibody or functional fragment thereof, wherein the antibody or functional fragment includes a variable heavy (VH) chain domain having the CDR1, CDR2 and CDR3 amino acid sequence of the clonal isolate 5B1, 9H3, 5H11 or 7E3. Such VH domains can include the amino acid residues 55-62, 70-77 and 116-131 of SEQ ID NO: 2, or alternatively the amino acid residues 45-52, 70-77 and 116-131 of SEQ ID NO: 6, or alternatively the amino acid residues 45-52, 70-77 and 116-131 of SEQ ID NO: 10, or alternatively the amino acid residues 45-52, 70-77 and 116-134 of SEQ ID NO: 14. In another aspect, the nucleotide sequence encoding the CDR1, CDR2 and CDR3 of the VH domain can respectively include the nucleotide sequence of residues 133-156, 208-231 and 346-393 of SEQ ID NO: 1, or alternatively the nucleotide sequence of residues 133-156, 208-231 and 346-393 of SEQ ID NO: 5, or alternatively the nucleotide sequence of residues 133-156, 208-231 and 346-393 of SEQ ID NO: 9, or alternatively the nucleotide sequence of residues 133-156, 208-231, 346-402 of SEQ ID NO: 13.

In another embodiment, the invention provides an isolated polynucleotide encoding an antibody or functional fragment thereof, wherein the antibody includes a variable light (VL) chain domain having the CDR1, CDR2 and CDR3 amino acid sequence of the clonal isolate 5B1, 9H3, 5H11 or 7E3. Such VL domain can include the amino acid residues 45-52, 70-72 and 109-120 of SEQ ID NO: 4, or alternatively the amino acid residues 45-52, 70-72 and 109-119 of SEQ ID NO: 8, or alternatively the amino acid residues 45-52, 70-72 and 109-120 of SEQ ID NO: 12, or alternatively the amino acid residues 49-53, 72-74 and 111-120 of SEQ ID NO: 16. In another aspect, the nucleotide sequence encoding the CDR1, CDR2 and CDR3 of the VH domain can respectively include the nucleotide sequence of residues 133-156, 208-216 and 325-360 of SEQ ID NO: 3, or alternatively the nucleotide sequence of residues 133-156, 208-216 and 325-357 of SEQ ID NO: 7, or alternatively the nucleotide sequence of residues 134-156, 208-216 and 325-360 of SEQ ID NO: 11, or alternatively the nucleotide sequence of residues 145-162, 214-222 and 331-360 of SEQ ID NO: 15

In another embodiment, the invention provides a variant of the polynucleotides provided herein. A variant when used in reference to a polynucleotide includes a polynucleotide having one or more modified nucleotides, such as, but not limited to, a methylated nucleotide or a nucleotide analog. Additionally, a variant polynucleotide can include a polynucleotide that is interrupted by non-nucleotide components. Modifications to a polynucleotide can be imparted before or after assembly of the polynucleotide using methods well known to those skilled in the art. For example, a polynucleotide can be modified after polymerization by conjugation with a labeling component using either enzymatic or chemical techniques (e.g., as described in Gottfried and Weinhold, 2011, Biochem. Soc. Trans., 39(2):523-628; Paredes et al., 2011, Methods, 54(2):251-259).

The polynucleotides can be obtained, and the nucleotide sequence of the polynucleotides determined, by any method well known in the art. Since the amino acid sequences of the variable heavy and light chain domains of 5B1, 9H3, 5H11 and 7E3 are known (see, e.g., SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14 and 16), nucleotide sequences encoding antibodies and modified versions of these antibodies can be determined using methods well known in the art, i.e., nucleotide codons known to encode particular amino acids are assembled in such a way to generate a nucleic acid that encodes the antibody. Such a polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, fragments, or variants thereof, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

A polynucleotide encoding an antibody or a functional fragment thereof of the invention can be generated using the nucleic acid sequence of the variable heavy and/or light chain domains of isolates 5B1, 9H3, 5H11 or 7E3 (e.g., SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13 and 15). A nucleic acid encoding the antibody or functional fragment can be chemically synthesized or obtained from a suitable source (e.g., cDNA isolated from cells expressing the antibody or functional fragment thereof, such as hybridoma cells selected to express the antibody or functional fragment thereof) by PCR amplification using synthetic primers hybridizable to the 3′ and 5′ ends of the sequence or by cloning using an oligonucleotide probe specific for the particular nucleic acid sequence. Amplified nucleic acids generated by PCR can then be cloned into replicable cloning vectors using any method well known in the art.

In some embodiments, the present invention provides an isolated antibody or functional fragment thereof, wherein the antibody binds to sLe^(a). Accordingly, in some aspects, the invention provides an isolated antibody or functional fragment thereof that binds to sLe^(a), wherein the antibody or functional fragment thereof includes a VH domain having an amino acid sequence selected from the group consisting of residues 20-142 of SEQ ID NO: 2, residues 20-142 of SEQ ID NO: 6, residues 20-142 of SEQ ID NO: 10, and residues 20-145 of SEQ ID NO: 14.

In some embodiments, the invention provides an isolated antibody or functional fragment thereof that binds to sLe^(a), wherein the antibody or functional fragment thereof includes a VL domain having an amino acid sequence selected from the group consisting of residues 20-130 of SEQ ID NO: 4, residues 20-129 of SEQ ID NO: 8, residues 20-130 of SEQ ID NO: 12, and residues 23-130 of SEQ ID NO: 16.

In some embodiments, the invention provides an isolated antibody or functional fragment thereof that binds to sLe^(a), wherein the antibody or functional fragment thereof includes both a VH domain and a VL domain, where the VH domain and the VL domain respectively include an amino acid sequence selected from the group consisting of residues 20-142 of SEQ ID NO: 2 and residues 20-130 of SEQ ID NO: 4; residues 20-142 of SEQ ID NO: 6 and residues 20-129 of SEQ ID NO: 8; residues 20-142 of SEQ ID NO: 10 and residues 20-130 of SEQ ID NO: 12; and residues 20-145 of SEQ ID NO: 14 and residues 23-130 of SEQ ID NO: 16.

In some embodiments, in order to bind sLe^(a), the antibody or functional fragment thereof of the invention has one or more of the CDRs depicted in FIGS. 1-8 or listed in Table 2. An antibody or functional fragment thereof that includes one or more of the CDRs, in particular CDR3, can specifically bind to sLe^(a) as described herein. Specific binding to sLe^(a) can include the specificity and affinity as provided in Example I for any of the antibodies provided herein. In some aspects, an antibody or functional fragment thereof of the invention can include the CDC activity and/or ADCC activity of any one of the clonal isolates 5B1, 9H3, 5H11 or 7E3 described herein.

In some embodiments, the invention provides an isolated antibody or functional fragment thereof, wherein the antibody includes a VH chain domain having the CDR1, CDR2 and CDR3 amino acid sequence of the clonal isolate 5B1, 9H3, 5H11 or 7E3. Such VH domains can include the amino acid residues 55-62, 70-77 and 116-131 of SEQ ID NO: 2, or alternatively the amino acid residues 45-52, 70-77 and 116-131 of SEQ ID NO: 6, or alternatively the amino acid residues 45-52, 70-77 and 116-131 of SEQ ID NO: 10, or alternatively the amino acid residues 45-52, 70-77 and 116-134 of SEQ ID NO: 14.

In some embodiments, the invention provides an isolated antibody or functional fragment thereof, wherein the antibody includes a VL chain domain having the CDR1, CDR2 and CDR3 amino acid sequence of the clonal isolate 5B1, 9H3, 5H11 or 7E3. Such VL domain can include the amino acid residues 45-52, 70-72 and 109-120 of SEQ ID NO: 4, or alternatively the amino acid residues 45-52, 70-72 and 109-119 of SEQ ID NO: 8, or alternatively the amino acid residues 45-52, 70-72 and 109-120 of SEQ ID NO: 12, or alternatively the amino acid residues 49-53, 72-74 and 111-120 of SEQ ID NO: 16.

In some aspects of the invention, the isolated antibody or functional fragment thereof is a monoclonal antibody. In some aspects of the invention, the isolated antibody or functional fragment thereof provided herein is an IgG or IgM isotype. In a further aspect of the invention, the antibody or function fragment thereof is an antibody of the IgG1 subclass.

In some embodiments, the antibody functional fragment of the invention can be, but is not limited to, a Fab, a Fab′, a F(ab′)₂, a Fabc, a scFV, a diabody, a triabody, minibody or a single-domain antibody (sdAB). In some aspects, the invention provides a diabody that includes the amino acid sequence of SEQ ID NO: 18 or 20. Such diabodies of the invention can be, in some aspects, encoded by a polynucleotide having the nucleic acid sequence of SEQ ID NO: 17 or 19. With respect to antibodies and functional fragments thereof, various forms, alterations and modifications are well known in the art. The sLe^(a) specific antibody fragments of the invention can include any of such various antibody forms, alterations and modifications. Examples of such various forms and terms as they are known in the art are set forth below.

In some embodiments, the invention provides a method of producing an antibody or functional fragment thereof of the invention. The method of the invention can include introducing a polynucleotide of the invention into a host cell, culturing the host cell under conditions and for a sufficient period of time to produce the encoded heavy and/or light chain of an antibody or functional fragment of the invention, and purifying the heavy and/or light chain of an antibody or functional fragment.

Recombinant expression of an antibody or functional fragment thereof of the invention that binds to a sLe^(a) antigen can include construction of an expression vector containing a polynucleotide that encodes the heavy and/or light chain of an antibody or functional fragment of the invention. Once a polynucleotide encoding an antibody or functional fragment thereof (preferably, but not necessarily, containing the heavy and/or light chain variable domain) of the invention has been obtained, the vector for the production of the antibody or functional fragment can be produced by recombinant DNA technology using techniques well known in the art. Methods for preparing a protein by expressing a polynucleotide containing an antibody or a functional fragment thereof encoding nucleotide sequence are described herein.

Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody or functional fragments thereof coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, thus, provides replicable vectors including a nucleotide sequence encoding an antibody or functional fragment thereof of the invention operably linked to a promoter. Such vectors can include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., International Publication Nos. WO 86/05807 and WO 89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of the antibody can be cloned into such a vector for expression of the entire heavy, the entire light chain, or both the entire heavy and light chains.

The expression vector can be transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody or functional fragment thereof of the invention. Thus, the invention includes host cells containing a polynucleotide encoding an antibody or functional fragment thereof of the invention operably linked to a heterologous promoter. In some embodiments for the expression of double-chained antibodies, vectors encoding both the heavy and light chains can be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.

A variety of host-expression vector systems can be utilized to express the antibody or functional fragments thereof of the invention (see, e.g., U.S. Pat. No. 5,807,715). Such host-expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently purified, but also represent cells which can, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, NS0, and 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). In some aspects, bacterial cells such as Escherichia coli, or eukaryotic cells, especially for the expression of whole recombinant antibody, are used for the expression of a recombinant antibody or functional fragment. For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., 1986, Gene 45:101; and Cockett et al., 1990, Bio/Technology 8:2). In some embodiments, antibodies or fragments thereof of the invention are produced in CHO cells. In one embodiment, the expression of nucleotide sequences encoding antibodies or functional fragments thereof of the invention which bind to sLe^(a) is regulated by a constitutive promoter, inducible promoter or tissue specific promoter.

In bacterial systems, a number of expression vectors can be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such an antibody is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified can be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO 12:1791), in which the antibody coding sequence can be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the like. pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody or functional fragment coding sequence can be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems can be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest can be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts (e.g., see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 8 1:355-359). Specific initiation signals can also be used for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bittner et al., 1987, Methods in Enzymol. 153:51-544).

In addition, a host cell strain can be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the antibody or functional fragment. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7030 and HsS78Bst cells.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the antibody or functional fragment of the invention can be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells can be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method can advantageously be used to engineer cell lines which express the antibody molecule.

A number of selection systems can be used, including but not limited to, the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthineguanine phosphoribosyltransferase (Szybalska & Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:8-17) genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA. 77(6):3567-70; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); glutamine synthetase (GS), which is an enzyme responsible for the biosynthesis of glutamine using glutamate and ammonia (Bebbington et al., 1992, Biuotechnology 10:169); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIB TECH 11(5):155-215); and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147). Methods well known in the art of recombinant DNA technology can be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, N Y (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, N Y (1990); and in Chapters 12 and 13, Dracopoli et al. (eds.), Current Protocols in Human Genetics, John Wiley & Sons, N Y (1994); Colberre-Garapin et al., 1981, J Mol. Biol. 150:1, which are incorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987)). When a marker in the vector system expressing an antibody or functional fragment thereof is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al., 1983, Mol. Cell. Biol. 3:257).

The host cell can be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors can contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector can be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain can be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature 322:52; and Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197-2199). The coding sequences for the heavy and light chains can include cDNA or genomic DNA.

Additionally, polynucleotides encoding the heavy and/or light chains of the antibody or functional fragment of the invention can be subjected to codon optimization using techniques well known in the art to achieve optimized expression of an antibody or functional fragment of the invention in a desired host cell. For example, in one method of codon optimization, a native codon is substituted by the most frequent codon from a reference set of genes, wherein the rate of codon translation for each amino acid is designed to be high. Additional exemplary methods for generating codon optimized polynucleotides for expression of a desired protein, which can be applied to the heavy and/or light chains of the antibody or functional fragment of the invention, are described in Kanaya et al., Gene, 238:143-155 (1999), Wang et al., Mol. Biol. Evol., 18(5):792-800 (2001), U.S. Pat. No. 5,795,737, U.S. Publication 2008/0076161 and WO 2008/000632.

Once an antibody molecule of the invention has been produced by recombinant expression, it can be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Further, the antibodies or functional fragments of the present invention can be fused to heterologous polypeptide sequences provided herein or otherwise known in the art to facilitate purification. For example, an antibody or functional fragment of the invention can be purified through recombinantly adding a poly-histidine tag (His-tag), FLAG-tag, hemagglutinin tag (HA-tag) or myc-tag among others that are commercially available and utilizing purification methods well known to those skilled in the art.

A Fab fragment refers to a monovalent fragment consisting of the VL, VH, CL and CH1 domains; a F(ab′)₂ fragment is a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consists of the VH and CH1 domains; an Fv fragment consists of the VL and VH domains of a single arm of an antibody; and a dAb fragment (Ward et al., Nature 341:544-546, (1989)) consists of a VH domain.

An antibody can have one or more binding sites. If there is more than one binding site, the binding sites can be identical to one another or can be different. For example, a naturally occurring immunoglobulin has two identical binding sites, a single-chain antibody or Fab fragment has one binding site, while a “bispecific” or “bifunctional” antibody has two different binding sites.

A single-chain antibody (scFv) refers to an antibody in which a VL and a VH region are joined via a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous polypeptide chain wherein the linker is long enough to allow the protein chain to fold back on itself and form a monovalent antigen binding site (see, e.g., Bird et al., Science 242:423-26 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-83 (1988)). Diabodies refer to bivalent antibodies including two polypeptide chains, wherein each polypeptide chain includes VH and VL domains joined by a linker that is too short to allow for pairing between two domains on the same chain, thus allowing each domain to pair with a complementary domain on another polypeptide chain (see, e.g., Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-48 (1993), and Poljak et al., Structure 2:1121-23 (1994)). If the two polypeptide chains of a diabody are identical, then a diabody resulting from their pairing will have two identical antigen binding sites. Polypeptide chains having different sequences can be used to make a diabody with two different antigen binding sites. Similarly, tribodies and tetrabodies are antibodies including three and four polypeptide chains, respectively, and forming three and four antigen binding sites, respectively, which can be the same or different.

The present invention also provides an antibody or functional fragment thereof derivative of 5B1, 9H3, 5H11 and/or 7E3, wherein the antibody or functional fragment binds to sLe^(a). Standard techniques well known to those of skill in the art can be used to introduce mutations in the nucleotide sequence encoding an antibody or functional fragment thereof of the invention, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis which results in amino acid substitutions. In some aspects, the derivative includes less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the original molecule.

In some embodiments, the invention provides an antibody or functional fragment thereof having modified forms of naturally occurring amino acids, conservative substitutions, non-naturally occurring amino acids, amino acid analogues and mimetics so long as such the antibody or functional fragment retains functional activity as defined herein. In one embodiment, the derivative has conservative amino acid substitutions that are made at one or more predicted non-essential amino acid residues. A conservative amino acid substitution is one in which the amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded antibody or functional fragment thereof can be expressed and the activity of the antibody or functional fragment can be determined.

In some embodiments, the invention provides an antibody or functional fragment thereof having modified fucosylation, galactosylation and/or sialylation of an Fc fragment contained within an antibody or functional fragment of the invention. Such modifications of an Fc fragment can effect Fc receptor-mediated activity as discussed in Peipp et al., Blood, 112(6):2390-2399 (2008). For example, glycoengineered therapeutic antibodies lacking core fucose residues from the Fc N-glycans exhibit strong ADCC at lower concentrations with much higher efficacy compared to fucosylated counterparts. Shields et al., J Biol. Chem., 277(30):26733-40 (2002); Okazaki et al., J Mol Biol., 336:1239-1249 (2004); Natsume et al., J. Immunol. Methods., 306:93-103 (2005). Methods for modifying the fucosylation, galactosylation and/or sialylation of an antibody for functional fragment thereof are well known in the art. For example, defucosylation approaches can be grouped into three methodologies (1) conversion of the N-glycosylation pathway of nonmammalian cells to the ‘humanized’ non-fucosylation pathway; (2) inactivation of the N-glycan fucosylation pathway of mammalian cells and (3) in vitro chemical synthesis of non-fucosylated N-glycoprotein or enzymatic modification of N-glycans to non-fucosylated forms, as described in Yamane-Ohnuki et al., MAbs., 1(3):230-236 (2009). It is understood that any one of these methods or any other method that is well known in the art can be used to produce an antibody or functional fragment thereof having modified fucosylation, galactosylation and/or sialylation.

Antibodies or functional fragments thereof of the invention that bind to sLe^(a) can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or by recombinant expression techniques. The practice of the invention employs, unless otherwise indicated, conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the skill of the art. These techniques are described in the references cited herein and are fully explained in the literature. See, e.g., Maniatis et al. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annual updates); Current Protocols in Immunology, John Wiley & Sons (1987 and annual updates) Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren et al. (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Borrebaeck (ed.) (1995) Antibody Engineering, Second Edition, Oxford University Press; Lo (ed.) (2006) Antibody Engineering: Methods and Protocols (Methods in Molecular Biology); Vol. 248, Humana Press, Inc; each of which is incorporated herein by reference in its entirety.

Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma and recombinant technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, N.Y., 1981), each of which is incorporated herein by reference in its entirety. A monoclonal antibody is not limited to antibodies produced through hybridoma technology. Other exemplary methods of producing monoclonal antibodies are known in the art. Additional exemplary methods of producing monoclonal antibodies are provided in Example I herein.

Antibody functional fragments which bind sLe^(a) can be generated by any technique well known to those of skill in the art. For example, Fab and F(ab′)₂ fragments of the invention can be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)₂ fragments). F(ab′)₂ fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain.

The antibody functional fragments of the invention can also be generated using various phage display methods known in the art. For example, in phage display methods, functional antibody domains, such as the heavy and/or light chain variable regions having one, two, three, four, five or six CDRs provided herein, are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. The DNA encoding the VH and VL domains are recombined together with an scFv linker by PCR and cloned into a phagemid vector. The vector is electroporated in E. coli and the E. coli is infected with helper phage. Phage used in these methods are typically filamentous phage including fd and M13 and the VH and VL domains are usually recombinantly fused to either the phage gene III or gene VIII. Phage expressing an antigen binding domain that binds to a particular antigen, such as sLe^(a), can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Examples of phage display methods that can be used to make the antibody functional fragments of the present invention include those disclosed in Brinkman et al., 1995, J. Immunol. Methods 182:41-50; Ames et al., 1995, J. Immunol. Methods 184:177-186; Kettleborough et al., 1994, Eur. J. Immunol. 24:952-958; Persic et al., 1997, Gene 187:9-18; Burton et al., 1994, Advances in Immunology 57:191-280; PCT Application No. PCT/GB91/01134; International Publication Nos. WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO 95/15982, WO 95/20401, and WO97/13844; and U.S. Pat. Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.

As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described herein.

Techniques to recombinantly produce Fab, Fab′ and F(ab′)₂ fragments can also be employed using methods known in the art such as those disclosed in PCT publication No. WO 92/22324; Mullinax et al., 1992, BioTechniques 12(6):864-869; Sawai et al., 1995, AJRI 34:26-34; and Better et al., 1988, Science 240:1041-1043, each of which is incorporated by reference in its entirety.

To generate whole antibodies, PCR primers including VH or VL nucleotide sequences, a restriction site, and a flanking sequence to protect the restriction site can be used to amplify the VH or VL sequences in scFv clones. Utilizing cloning techniques well known to those of skill in the art, the PCR amplified VH domains can be cloned into vectors expressing a VH constant region, e.g., the human gamma 1 constant region, and the PCR amplified VL domains can be cloned into vectors expressing a VL constant region, e.g., human kappa or lambda constant regions. The VH and VL domains can also be cloned into one vector expressing the necessary constant regions. The heavy chain conversion vectors and light chain conversion vectors are then co-transfected into cell lines to generate stable or transient cell lines that express full-length antibodies, e.g., IgG, using techniques well known to those of skill in the art.

In some embodiments, an antibody or functional fragment of the invention is conjugated (covalent or non-covalent conjugations) or recombinantly fused to one or more diagnostic agent, detectable agent or therapeutic agent or any other desired molecule. The conjugated or recombinantly fused antibody or functional fragment can be useful for monitoring or diagnosing the onset, development, progression and/or severity of a disease associated with the expression of sLe^(a), such as cancer or tumor formation, as part of a clinical testing procedure, such as determining the efficacy of a particular therapy.

Detection and diagnosis can be accomplished, for example, by coupling the antibody or functional fragment of the invention to detectable substances including, but not limited to, radioactive materials, such as, but not limited to, zirconium (⁸⁹Zr), iodine (¹³¹I, ¹²⁵I, ¹²⁴I, ¹²³I, and ¹²¹I), carbon (¹⁴C, ¹¹C) sulfur (³⁵S), tritium (³H), indium (¹¹⁵In, ¹¹³In, ¹¹²In, and ¹¹¹In), technetium (⁹⁹Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F) ¹⁵O, ¹³N, ⁶⁴Cu, ^(94m)Tc, ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁸⁶Y, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn, and ¹¹⁷Sn; and positron emitting metals using various positron emission tomographies, various enzymes, such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as, but not limited to, streptavidin/biotin and avidin/biotin; fluorescent materials, such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials, such as, but not limited to, luminol; bioluminescent materials, such as but not limited to, luciferase, luciferin, and aequorin, and non-radioactive paramagnetic metal ions.

The present invention further encompasses therapeutic uses of an antibody or functional fragment of the invention conjugated (covalent or non-covalent conjugations) or recombinantly fused to one or more therapeutic agent. In this context, for example, the antibody may be conjugated or recombinantly fused to a therapeutic agent, such as a cytotoxin, e.g., a cytostatic or cytocidal agent, or a radioactive metal ion, e.g., alpha-emitters. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. A therapeutic agent can be a chemotherapeutic such as, but is not limited to, an anthracycline (e.g., doxorubicin and daunorubicin (formerly daunomycin)); a taxan (e.g., paclitaxel (Taxol) and docetaxel (Taxotere); an antimetabolite (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil and decarbazine); or an alkylating agent (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BCNU), lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cisdichlorodiamine platinum (II) (DDP) and cisplatin); an antibiotic (e.g., actinomycin D, bleomycin, mithramycin, and anthramycin (AMC)); an Auristatin molecule (e.g., auristatin PHE, bryostatin 1, solastatin 10, monomethyl auristatin E (MMAE) and monomethylauristatin F (MMAF)); a hormone (e.g., glucocorticoids, progestins, androgens, and estrogens); a nucleoside analoge (e.g. Gemcitabine), a DNA-repair enzyme inhibitor (e.g., etoposide and topotecan), a kinase inhibitor (e.g., compound ST1571, also known as Gleevec or imatinib mesylate); a cytotoxic agent (e.g., maytansine, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, 1-dehydrotestosterone, glucorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and analogs or homologs thereof, and those compounds disclosed in U.S. Pat. Nos. 6,245,759, 6,399,633, 6,383,790, 6,335,156, 6,271,242, 6,242,196, 6,218,410, 6,218,372, 6,057,300, 6,034,053, 5,985,877, 5,958,769, 5,925,376, 5,922,844, 5,911,995, 5,872,223, 5,863,904, 5,840,745, 5,728,868, 5,648,239, 5,587,459); a farnesyl transferase inhibitor (e.g., R115777, BMS-214662, and those disclosed by, for example, U.S. Pat. Nos. 6,458,935, 6,451,812, 6,440,974, 6,436,960, 6,432,959, 6,420,387, 6,414,145, 6,410,541, 6,410,539, 6,403,581, 6,399,615, 6,387,905, 6,372,747, 6,369,034, 6,362,188, 6,342,765, 6,342,487, 6,300,501, 6,268,363, 6,265,422, 6,248,756, 6,239,140, 6,232,338, 6,228,865, 6,228,856, 6,225,322, 6,218,406, 6,211,193, 6,187,786, 6,169,096, 6,159,984, 6,143,766, 6,133,303, 6,127,366, 6,124,465, 6,124,295, 6,103,723, 6,093,737, 6,090,948, 6,080,870, 6,077,853, 6,071,935, 6,066,738, 6,063,930, 6,054,466, 6,051,582, 6,051,574, and 6,040,305); a topoisomerase inhibitor (e.g., camptothecin, irinotecan, SN-38, topotecan, 9-aminocamptothecin, GG-211 (GI 147211), DX-8951f, IST-622, rubitecan, pyrazoloacridine) CR-5000, saintopin, UCE6, UCE1022, TAN-1518A, TAN 1518B, KT6006, KT6528, ED-110, NB-506, ED-110, NB-506, fagaronine, coralyne, beta-lapachone and rebeccamycin); a DNA minor groove binder (e.g., Hoescht dye 33342 and Hoechst dye 33258); adenosine deaminase inhibitors (e.g., Fludarabine phosphate and 2-Chlorodeoxyadenosine); or pharmaceutically acceptable salts, solvates, clathrates, or prodrugs thereof. A therapeutic agent can be a immunotherapeutic such as, but is not limited to, cetuximab, bevacizumab, heceptin, rituximab).

In addition, an antibody or functional fragment of the invention can be conjugated to a therapeutic agent such as a radioactive metal ion, such as alpha-emitters such as ²¹³Bi or macrocyclic chelators useful for conjugating radiometal ions, including but not limited to, ¹³¹In, ¹³¹LU, ¹³¹Y, ¹³¹Ho, ¹³¹Sm; or a macrocyclic chelator, such as 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid (DOTA) which can be attached to the antibody or functional fragment via a linker molecule. Such linker molecules are commonly known in the art and described in Denardo et al., 1998, Clin Cancer Res. 4(10):2483-90; Peterson et al., 1999, Bioconjug. Chem. 10(4):553-7; and Zimmerman et al., 1999, Nucl. Med. Biol. 26(8):943-50.

Further, an antibody or functional fragment of the invention may be conjugated (covalent or non-covalent conjugations) or recombinantly fused to a therapeutic agent that modifies a given biological response. Thus, therapeutic agents are not to be construed as limited to classical chemical therapeutic agents. For example, the therapeutic agent can be a protein, peptide, or polypeptide possessing a desired biological activity. Such proteins can include, for example, a toxin (e.g., abrin, ricin A, pseudomonas exotoxin, cholera toxin and diphtheria toxin); a protein such as tumor necrosis factor, γ-interferon, α-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent (e.g., TNF-γ, AIM I, AIM II, Fas Ligand and VEGF), an anti-angiogenic agent (e.g., angiostatin, endostatin and a component of the coagulation pathway such as tissue factor); a biological response modifier (e.g., a cytokine such as interferon gamma, interleukin-1, interleukin-2, interleukin-5, interleukin-6, interleukin-7, interleukin-9, interleukin-10, interleukin-12, interleukin-15, interleukin-23, granulocyte macrophage colony stimulating factor, and granulocyte colony stimulating factor); a growth factor (e.g., growth hormone), or a coagulation agent (e.g., calcium, vitamin K, tissue factors, such as but not limited to, Hageman factor (factor XII), high-molecular-weight kininogen (HMWK), prekallikrein (PK), coagulation proteins-factors II (prothrombin), factor V, XIIa, VIII, XIIIa, XI, XIa, IX, IXa, X, phospholipid, and fibrin monomer).

The present invention encompasses antibodies or functional fragments of the invention recombinantly fused or chemically conjugated (covalent or non-covalent conjugations) to a heterologous protein or polypeptide to generate fusion proteins. In some aspects, such a polypeptide can be about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90 or about 100 amino acids in length. In some aspects, the invention provides fusion proteins having a functional fragment of an antibody of the invention (e.g., a Fab fragment, Fd fragment, Fv fragment, F(ab)₂ fragment, a VH domain, a VH CDR, a VL domain or a VL CDR) and a heterologous protein or polypeptide. In one embodiment, the heterologous protein or polypeptide that the antibody or functional fragment is fused to is useful for targeting the antibody or functional fragment to a particular cell type, such as a cell that expresses sLe^(a).

A conjugated or fusion protein of the invention includes any antibody or functional fragment of the invention provided herein conjugated (covalent or non-covalent conjugations) or recombinantly fused to a diagnostic agent, detectable agent or therapeutic agent. In one embodiment, a conjugated or fusion protein of the invention includes a 5B1, 9H3, 5H11 or 7E3 antibody, and a diagnostic agent, detectable agent or therapeutic agent. In another embodiment, a conjugated or fusion protein of the invention includes a functional fragment of 5B1, 9H3, 5H11 or 7E3 antibodies, and a diagnostic agent, detectable agent or therapeutic agent. In another embodiment, a conjugated or fusion protein of the invention includes a VH domain having the amino acid sequence of any one of the VH domains depicted in residues 20-142 of SEQ ID NO: 2, residues 20-142 of SEQ ID NO: 6, residues 20-142 of SEQ ID NO: 10, or residues 20-145 of SEQ ID NO: 14, and/or a VL domain having the amino acid sequence of any one of the VL domains depicted in residues 20-130 of SEQ ID NO: 4, residues 20-129 of SEQ ID NO: 8, residues 20-130 of SEQ ID NO: 12, or residues 23-130 of SEQ ID NO: 16, and a diagnostic agent, detectable agent or therapeutic agent. In another embodiment, a conjugated or fusion protein of the present invention includes one or more VH CDRs having the amino acid sequence of any one of the VH CDRs depicted in SEQ ID NOS: 2, 6, 10 or 14, and a diagnostic agent, detectable agent or therapeutic agent. In another embodiment, a conjugated or fusion protein includes one or more VL CDRs having the amino acid sequence of any one of the VL CDRs depicted in SEQ ID NOS: 4, 8, 12 or 16, and a diagnostic agent, detectable agent or therapeutic agent. In another embodiment, a conjugated or fusion protein of the invention includes at least one VH domain and at least one VL domain depicted in residues 20-142 of SEQ ID NO: 2 and residues 20-130 of SEQ ID NO: 4; residues 20-142 of SEQ ID NO: 6 and residues 20-129 of SEQ ID NO: 8; residues 20-142 of SEQ ID NO: 10 and residues 20-130 of SEQ ID NO: 12; or residues 20-145 of SEQ ID NO: 14 and residues 23-130 of SEQ ID NO: 16, respectively, and a diagnostic agent, detectable agent or therapeutic agent.

Methods for fusing or conjugating diagnostic agents, detectable agents or therapeutic agents (including polypeptides) to antibodies are well known, see, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies 84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), Thorpe et al., 1982, Immunol. Rev. 62:119-58; U.S. Pat. Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,723,125, 5,783,181, 5,908,626, 5,844,095, 5,112,946, 7,981,695, 8,039,273, 8,142,784; U.S. Publications 2009/0202536, 2010/0034837, 2011/0137017, 2011/0280891, 2012/0003247; EP 307,434; EP 367,166; EP 394,827; PCT publications WO 91/06570, WO 96/04388, WO 96/22024, WO 97/34631, and WO 99/04813; Ashkenazi et al., Proc. Natl. Acad. Sci. USA, 88: 10535-10539, 1991; Traunecker et al., Nature, 331:84-86, 1988; Zheng et al., J. Immunol., 154:5590-5600, 1995; Vil et al., Proc. Natl. Acad. Sci. USA, 89:11337-11341, 1992; and Senter, Current Opinion in Chemical Biology, 13:235-244 (2009), which are incorporated herein by reference in their entireties.

In another aspect, a diagnostic agent, detectable agent or therapeutic agent can be attached at the hinge region of a reduced antibody component via disulfide bond formation. Alternatively, such agents can be attached to the antibody component using a heterobifunctional cross-linker, such as N-succinyl 3-(2-pyridyldithio)proprionate (SPDP). Yu et al., Int. J. Cancer 56: 244 (1994). General techniques for such conjugation are well known in the art. See, for example, Wong, CHEMISTRY OF PROTEIN CONJUGATION AND CROSS-LINKING (CRC Press 1991); Upeslacis et al., “Modification of Antibodies by Chemical Methods,” in MONOCLONAL ANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc. 1995); Price, “Production and Characterization of Synthetic Peptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.), pages 60-84 (Cambridge University Press 1995).

Alternatively, a diagnostic agent, detectable agent or therapeutic agent can be conjugated via a carbohydrate moiety in the Fc region of the antibody. Methods for conjugating peptides to antibody components via an antibody carbohydrate moiety are well known to those of skill in the art. See, for example, Shih et al., Int. J. Cancer. 41:832-839 (1988); Shih et al., Int. J. Cancer. 46:1101-1106 (1990); and Shih et al., U.S. Pat. No. 5,057,313, all of which are incorporated in their entirety by reference. The general method involves reacting an antibody component having an oxidized carbohydrate portion with a carrier polymer that has at least one free amine function and that is loaded with a plurality of peptide. This reaction results in an initial Schiff base (imine) linkage, which can be stabilized by reduction to a secondary amine to form the final conjugate.

However, if the Fc region is absent, for example, if an antibody functional fragment as provided herein is desirable, it is still possible to attach a diagnostic agent, a detectable agent or a therapeutic agent. A carbohydrate moiety can be introduced into the light chain variable region of a full-length antibody or antibody fragment. See, for example, Leung et al., J. Immunol., 154: 5919 (1995); U.S. Pat. Nos. 5,443,953 and 6,254,868, all of which are incorporated in their entirety by reference. The engineered carbohydrate moiety is used to attach the diagnostic agent, detectable agent or therapeutic agent.

The therapeutic agent conjugated or recombinantly fused to an antibody functional fragment of the invention that binds to sLe^(a) can be chosen to achieve the desired prophylactic or therapeutic effect(s). It is understood that it is within the skill level of a clinician or other medical personnel to consider the following when deciding which therapeutic agent to conjugate or recombinantly fuse to an antibody or functional fragment of the invention: the nature of the disease, the severity of the disease, and the condition of the subject.

A conjugate or fusion antibody or functional fragment of the invention that is detectably labeled as provided herein and binds to sLe^(a) can be used for diagnostic purposes to detect, diagnose, or monitor a disease, wherein the cells that cause or are associated with the disease express sLe^(a). For example, as provided herein, cancer cells and tumors have been shown to express sLe^(a), such as, but not limited to, tumors of the gastrointestinal tract, breast cancer, ovarian cancer, colon cancer, colorectal adenocarcinoma, pancreatic cancer, pancreatic adenocarcinoma, small cell carcinoma of the lung, bladder adenocarcinoma, metastatic colon cancer, colorectal cancer, signet ring ovarian cancer and metastatic carcinoma. Accordingly, the invention provides methods for detecting cancer or a tumor formation in a subject by administering an effective amount of a conjugate or fusion antibody or functional fragment of the invention to a subject in need thereof. In some aspects, the detection method can further include assaying the expression of a sLe^(a) on the cells or a tissue sample of a subject using one or more conjugates or fusion antibodies or functional fragments of the invention that bind to sLe^(a); and comparing the level of the sLe^(a) with a control level, e.g., levels in normal tissue samples (e.g., from a subject not having a disease, or from the same subject before disease onset), whereby an increase in the assayed level of sLe^(a) compared to the control level of the sLe^(a) is indicative of the disease. Such diagnostic methods can allow health professionals to employ preventative measures or aggressive treatment earlier than otherwise possible thereby preventing the development or further progression of the disease.

An antibody or functional fragment of the invention can also be used to assay sLe^(a) antigen levels in a biological sample using classical immunohistological methods as provided herein or as well known to those of skill in the art (e.g., see Jalkanen et al., 1985, J. Cell. Biol. 101:976-985; and Jalkanen et al., 1987, J. Cell. Biol. 105:3087-3096). Other antibody-based methods useful for detecting sLe^(a) include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (MA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (¹²⁵I, ¹²¹I) carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹²¹In), and technetium (⁹⁹Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.

In one aspect, the invention provides for the detection and diagnosis of disease in a human. In one embodiment, diagnosis includes: a) administering (for example, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a conjugate or fusion protein of the invention that binds to sLe^(a); b) waiting for a time interval following the administering for permitting the conjugate or fusion protein to preferentially concentrate at sites in the subject where sLe^(a) is expressed (and, in some aspects, for unbound conjugate or fusion protein to be cleared to background level); c) determining background level; and d) detecting the conjugate or fusion protein in the subject, such that detection of conjugate or fusion protein above the background level indicates that the subject has a disease. Background level can be determined by various methods including, comparing the amount of conjugate or fusion protein detected to a standard value previously determined for a particular system.

It is understood that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images and can be readily determined by one of skill in the art. For example, in the case of a radioisotope conjugated to an antibody or functional fragment of the invention, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of ⁹⁹Tc. The conjugate will then preferentially accumulate at the location of cells which express sLe^(a). In vivo tumor imaging is described in S. W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).

Depending on several variables, including the type of detectable agent used and the mode of administration, the time interval following the administration for permitting the conjugate to preferentially concentrate at sites in the subject and for unbound conjugate to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment, the time interval following administration is 5 to 20 days or 5 to 10 days. In one embodiment, monitoring of a disease is carried out by repeating the method for diagnosing as provided herein, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, or longer.

The presence of the conjugate or fusion protein can be detected in the subject using methods known in the art for in vivo scanning. These methods depend upon the type of detectable agent used. A skilled artisan will be able to determine the appropriate method for detecting a particular detectable agent. Methods and devices that may be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography. In one embodiment, an antibody or function fragment of the invention is conjugated to a radioisotope and is detected in the subject using a radiation responsive surgical instrument. In another embodiment, an antibody or function fragment of the invention is conjugated to a fluorescent compound and is detected in the subject using a fluorescence responsive scanning instrument. In another embodiment, an antibody or function fragment of the invention is conjugated to a positron emitting metal, such as zirconium (⁸⁹Zr) or any other positron emitting metal provided herein or that is well known in the art to be detectable by positron emission-tomography, and is detected in the subject using positron emission-tomography. In yet another embodiment, an antibody or function fragment of the invention is conjugated to a paramagnetic label and is detected in a subject using magnetic resonance imaging (MM).

In one embodiment, the invention provides a pharmaceutical composition having an antibody or a functional fragment of the invention and a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier that can be used in the pharmaceutical compositions of the invention include any of the standard pharmaceutical carriers known in the art, such as phosphate buffered saline solution, water and emulsions such as an oil and water emulsion, and various types of wetting agents. These pharmaceutical compositions can be prepared in liquid unit dose forms or any other dosing form that is sufficient for delivery of the antibody or functional fragment of the invention to the target area of the subject in need of treatment. For example, the pharmaceutical compositions can be prepared in any manner appropriate for the chosen mode of administration, e.g., intravascular, intramuscular, sub-cutaneous, intraperitoneal, etc. Other optional components, e.g., pharmaceutical grade stabilizers, buffers, preservatives, excipients and the like can be readily selected by one of skill in the art. The preparation of a pharmaceutically composition, having due regard to pH, isotonicity, stability and the like, is within the level of skill in the art.

Pharmaceutical formulations containing one or more antibodies or functional fragments of the invention provided herein can be prepared for storage by mixing the antibody having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

Thus, in some embodiments, the invention provides a method for treating or preventing a disease in a subject in need thereof. The methods of the invention can include administering a therapeutically effective amount of a pharmaceutical composition provided herein to the subject. For example, the pharmaceutical composition can include one or more antibody or functional fragment provided herein. Diseases that can be treated or prevented using the methods of the invention include cancer, tumor formation and/or metastasis. In particular, the methods of the invention are useful for treating cancers or tumor formation wherein the cancer cells or tumor expresses the carbohydrate sLe^(a). Non-limiting examples of cancers or tumors that can be treated or prevented using the methods of the invention include tumors of the gastrointestinal tract, for example, colon cancer, colorectal adenocarcinoma, metastatic colon cancer, colorectal cancer, pancreatic cancer, or pancreatic adenocarcinoma; small cell carcinoma of the lung; bladder adenocarcinoma; signet ring ovarian cancer; ovarian cancer, metastatic carcinoma; and adenocarcinoma of the stomach, esophagus, throat, urogenital tract, or breast.

Accordingly, in some aspects, the invention provides a method for treating cancer or preventing tumor metastasis in a subject in need thereof by administering a therapeutically effective amount of a pharmaceutical composition having an antibody or functional fragment thereof, wherein the antibody or functional fragment binds to sLe^(a) and includes a VH domain having an amino acid sequence selected from the group consisting of residues 20-142 of SEQ ID NO: 2, residues 20-142 of SEQ ID NO: 6, residues 20-142 of SEQ ID NO: 10, and residues 20-145 of SEQ ID NO: 14. In another aspect, the invention provides a method for treating cancer or preventing tumor metastasis in a subject in need thereof by administering a therapeutically effective amount of a pharmaceutical composition having an antibody or functional fragment thereof, wherein the antibody or functional fragment binds to sLe^(a) and includes a VL domain having an amino acid sequence selected from the group consisting of residues 20-130 of SEQ ID NO: 4, residues 20-129 of SEQ ID NO: 8, residues 20-130 of SEQ ID NO: 12, and residues 23-130 of SEQ ID NO: 16. In yet another aspect, the invention provides a method for treating cancer or preventing tumor metastasis in a subject in need thereof by administering a therapeutically effective amount of a pharmaceutical composition having an antibody or functional fragment thereof, wherein the antibody or functional fragment binds to sLe^(a) and includes both a VH domain and a VL domain, where the VH domain and the VL domain respectively include an amino acid sequence selected from the group consisting of residues 20-142 of SEQ ID NO: 2 and residues 20-130 of SEQ ID NO: 4; residues 20-142 of SEQ ID NO: 6 and residues 20-129 of SEQ ID NO: 8; residues 20-142 of SEQ ID NO: 10 and residues 20-130 of SEQ ID NO: 12; and residues 20-145 of SEQ ID NO: 14 and residues 23-130 of SEQ ID NO: 16.

Formulations, such as those described herein, can also contain more than one active compound as necessary for the particular disease being treated. In certain embodiments, formulations include an antibody or functional fragment of the invention and one or more active compounds with complementary activities that do not adversely affect each other. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. For example, an antibody or functional fragment of the invention can be combined with one or more other therapeutic agents. Such combined therapy can be administered to the subject concurrently or successively.

Thus, in some aspects, invention provides a method for treating or preventing a disease by administering a therapeutically effective amount of a pharmaceutical composition provided herein to a subject in need thereof, wherein the pharmaceutical composition includes an antibody or functional fragment of the invention and a second therapeutic agent. The appropriate second therapeutic agent can be readily determined by one of ordinary skill in the art as discussed herein. As provided herein in Example IV, in some aspects of the invention, the second therapeutic agent can be Taxol.

The pharmaceutical compositions provided herein contain therapeutically effective amounts of one or more of the antibodies of the invention provided herein, and optionally one or more additional therapeutic agents, in a pharmaceutically acceptable carrier. Such pharmaceutical compositions are useful in the prevention, treatment, management or amelioration of a disease, such cancer or tumor formation, or one or more of the symptoms thereof.

The pharmaceutical compositions can contain one or more antibodies or functional fragments of the invention. In one embodiment, the antibodies or functional fragments are formulated into suitable pharmaceutical preparations, such as sterile solutions or suspensions for parenteral administration. In one embodiment, the antibodies or functional fragments provided herein are formulated into pharmaceutical compositions using techniques and procedures well known in the art (see, e.g., Ansel (1985) Introduction to Pharmaceutical Dosage Forms, 4^(th) Ed., p. 126).

An antibody or functional fragment of the invention can be included in the pharmaceutical composition in a therapeutically effective amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the subject treated. The therapeutically effective concentration can be determined empirically by testing the compounds in in vitro and in vivo systems using routine methods and then extrapolated therefrom for dosages for humans. The concentration of an antibody or functional fragment in the pharmaceutical composition will depend on, e.g., the physicochemical characteristics of the antibody or functional fragment, the dosage schedule, and amount administered as well as other factors well known to those of skill in the art.

In one embodiment, a therapeutically effective dosage produces a serum concentration of an antibody or functional fragment of from about 0.1 ng/ml to about 50-100 μg/ml. The pharmaceutical compositions, in another embodiment, provide a dosage of from about 0.001 mg to about 500 mg of antibody per kilogram of body weight per day. Pharmaceutical dosage unit forms can be prepared to provide from about 0.01 mg, 0.1 mg or 1 mg to about 30 mg, 100 mg or 500 mg, and in one embodiment from about 10 mg to about 500 mg of the antibody or functional fragment and/or a combination of other optional essential ingredients per dosage unit form.

The antibody or functional fragment of the invention can be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and can be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values can also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens can be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.

Upon mixing or addition of the antibody or functional fragment of the invention, the resulting mixture can be a solution, suspension or the like. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. The effective concentration is sufficient for ameliorating the symptoms of the disease, disorder or condition treated and may be empirically determined.

The pharmaceutical compositions are provided for administration to humans and animals in unit dosage forms, such as sterile parenteral solutions or suspensions containing suitable quantities of the compounds or pharmaceutically acceptable derivatives thereof. The antibody or functional fragment can be, in one embodiment, formulated and administered in unit-dosage forms or multiple-dosage forms. Unit-dose forms refers to physically discrete units suitable for human and animal subjects and packaged individually as is known in the art. Each unit-dose contains a predetermined quantity of the antibody or functional fragment of the invention sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical carrier, vehicle or diluent. Examples of unit-dose forms include ampoules and syringes. Unit-dose forms can be administered in fractions or multiples thereof. A multiple-dose form is a plurality of identical unit-dosage forms packaged in a single container to be administered in segregated unit-dose form. Examples of multiple-dose forms include vials or bottles of pints or gallons. Hence, multiple dose form is a multiple of unit-doses which are not segregated in packaging.

In one embodiment, one or more antibody or functional fragment of the invention is in a liquid pharmaceutical formulation. Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, or otherwise mixing an antibody or functional fragment as provided herein and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a solution. If desired, the pharmaceutical composition to be administered can also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents and the like, for example, acetate, sodium citrate, cyclodextrine derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA.

Methods for administering a pharmaceutical composition of the invention are well known in the art. It is understood that the appropriate route of administration of a pharmaceutical composition can be readily determined by a skilled clinician. Exemplary routes of administration include intravenous injection, intramuscular injection, intradermal injection or subcutaneous injection. Moreover, it is understood that the formulation of the pharmaceutical composition can be readily adjusted to accommodate the route of administration. The invention also provides that following administration of a pharmaceutical composition of the invention, delayed, successive and/or repeated dosages of one or more pharmaceutical composition as provided herein may be administered to the subject.

The methods of the invention for treating a disease is intended to include (1) preventing the disease, i.e., causing the clinical symptoms of the disease not to develop in a subject that may be predisposed to the disease but does not yet experience or display symptoms of the disease; (2) inhibiting the disease, i.e., arresting or reducing the development of the disease or its clinical symptoms; or (3) relieving the disease, i.e., causing regression of the disease or its clinical symptoms. The methods of the invention for preventing a disease is intended to include forestalling of a clinical symptom indicative of cancer or tumor formation. Such forestalling includes, for example, the maintenance of normal physiological indicators in a subject. Therefore, preventing can include the prophylactic treatment of a subject to guard them from the occurrence of tumor metastasis.

The therapeutically effective amount of the pharmaceutical composition used in the methods of the invention will vary depending on the pharmaceutical composition used, the disease and its severity and the age, weight, etc., of the subject to be treated, all of which is within the skill of the attending clinician. A subject that that can be treated by the methods of the invention include a vertebrate, preferably a mammal, more preferably a human.

It is understood that modifications which do not substantially affect the activity of the various embodiments of this invention are also provided within the definition of the invention provided herein. Accordingly, the following examples are intended to illustrate but not limit the present invention.

Example I Human Monoclonal Antibodies to sLe^(a) have Potent Antitumor Activity

The carbohydrate antigen sLe^(a) is widely expressed on epithelial tumors of the gastrointestinal tract, breast, and pancreas and on small-cell lung cancers. Since over-expression of sLe^(a) appears to be a key event in invasion and metastasis of many tumors and results in susceptibility to antibody-mediated lysis, sLe^(a) is an attractive molecular target for tumor therapy. Accordingly, as described herein, fully human monoclonal antibodies (mAb) from blood lymphocytes from individuals immunized with a sLe^(a)-KLH vaccine were generated and characterized. Several mAbs were selected based on ELISA and FACS including two mAbs with high affinity for sLe^(a) (5B1 and 7E3, binding affinities 0.14 and 0.04 nmol/L, respectively) and further characterized. Both antibodies were specific for Neu5Acα2-3Galβ1-3(Fucα1-4)G1cNAcβ and Neu5Gcα2-3Galβ1-3(Fucα1-4)G1cNAcβ as determined by glycan array analysis. Complement-dependent cytotoxicity against DMS-79 cells was higher (EC50 0.1 μg/mL vs. 1.7 μg/mL) for r7E3 (IgM) than for r5B1 (IgG1). In addition, r5B1 antibodies showed high level of antibody-dependent cell-mediated cytotoxicity activity on DMS-79 cells with human NK cells or peripheral blood mononuclear cells. To evaluate in vivo efficacy, the antibodies were tested in a xenograft model with Colo205 tumor cells or DMS-79 tumor cells engrafted into severe combined immunodeficient (SCID) mice. In the Colo205 xenograft model, treatment during the first 21 days with four doses of r5B1 (100 μg per dose) doubled the median survival time to 207 days, and three of five animals survived with six doses. In the DSM-79 xenograft model, growth of established DMS-79 tumors was suppressed or regressed in animals treated with r5B1 antibody. On the basis of the potential of sLe^(a) as a target for immune attack and their affinity, specificity, and effector functions, 5B1 and 7E3 have clinical utility in the treatment of cancer.

Materials, Cells, and Antibodies

DMS-79 (Pettengill et al., Cancer, 45:906-18 (1980)), SW626, EL4, HT29, BxPC3, SK-MEL28, and P3×63Ag8.653 cell lines were purchased from American Type Culture Collection (ATCC). Colo205-luc cells (Bioware ultra) were obtained from Caliper Life Sciences. The murine control mAb 121SLE (IgM) was purchased from GeneTex. sLe^(a) tetrasaccharide (Cat #S2279) was purchased from Sigma-Aldrich. sLe^(a)-HSA (human serum albumin) conjugate (Cat #07-011), monovalent biotinylated sLe^(a) (sLe^(a)-sp-biotin; Cat #02-044), polyvalent biotinylated sLe^(a)-PAA (Cat #01-044), biotin-labeled Le^(a)-PAA (Cat #01-035), and sLe^(x)-PAA-biotin (Cat #01-045) were purchased from GlycoTech. In the polyvalent presentation, the tetrasaccharide is incorporated into a polyacrylamide matrix (PAA), thereby creating a 30-kDa multivalent polymer with approximately every fifth amide group of the polymer chain N-substituted with biotin in a 4:1 ratio and approximately 20% carbohydrate content. Other HSA or BSA glycoconjugates used in this study were prepared in-house using sLe^(a) pentenyl glycoside as described. Ragupathi et al., Cancer Immunol Immunother, 58:1397-405 (2009). GD3, fucosyl-GM1, GM2, and GM3 were purchased from Matreya, and GD2 was purchased from Advanced ImmunoChemical.

Generation of Anti-sLe^(a) mAb-Producing Hybridomas

Blood samples were obtained from 3 patients in an ongoing trial with sLe^(a)-KLH conjugate vaccine in patients with breast cancer initiated at MSKCC under an MSKCC- and FDA-approved IRB protocol and IND. Blood specimens were selected from 2 patients after 3 or 4 vaccinations, which showed antibody titers of 1/160 and 1/320, respectively, against sLe^(a). These sera (and murine mAb 19.9) react well with sLe^(a)-positive cell lines in FACS assays and mediate potent CDC. Ragupathi et al., Cancer Immunol Immunother, 58:1397-405 (2009). Peripheral blood mononuclear cells (PBMC) were isolated from approximately 80 to 90 mL of blood by gradient centrifugation on Histopaque-1077 (Sigma-Aldrich).

PBMCs were cultured in RPMI-1640 medium supplemented with L-glutamine, nonessential amino acids, sodium pyruvate, vitamin, penicillin/streptomycin, 10% FBS (Omega Scientific), 10 ng/mL IL-21 (Biosource), and 1 μg/mL anti-CD40 mAb (G28-5 hybridoma supernatant; ATCC). Cells were fused by electrofusion to P3×63Ag8.653 myeloma cells.

sLe^(a) ELISA

For the sLe^(a) ELISA, plates were coated either with 1 μg/mL of sLe^(a)-HSA conjugate, monovalent biotinylated sLe^(a), or with polyvalent biotinylated sLe^(a)-PAA captured on Neutr-Avidin-coated plates. Uncoated wells (PBS) and HSA-coated wells were used as controls. Bound antibodies were initially detected with horseradish peroxidase (HRP)-labeled goat anti-human IgA+G+M (Jackson ImmunoResearch), and positive wells were subsequently probed with IgG-Fc- or IgM-specific secondary antibodies to determine isotypes.

Carbohydrate Specificity Analysis

Cross-reactivity against the closely related antigens, Le^(a) and sLe^(x), was evaluated by surface plasmon resonance (SPR) and confirmed by ELISA using biotin-labeled Le^(a)-PAA and biotin-sLe^(x)-PAA. Binding to gangliosides GD2, GD3, fucosyl-GM1, GM2, and GM3 was tested by ELISA. A competition ELISA was used to evaluate the specificity of the mAbs against several other related carbohydrate moieties. In brief, 2 μg/mL sLe^(a)-HSA conjugate was coated onto plates followed by blocking with 3% BSA in PBS. Next, 30 μL of different carbohydrate moieties (40 μg/mL in PBS prepared from 1 mg/mL stock solutions) either unconjugated or conjugated to HSA or BSA was mixed separately with 30 μL of test antibody and incubated at room temperature in a sample plate. After 30 minutes 50 μL of the mixture was transferred to the coated assay plate and incubated for 1 hour, followed by incubation with HRP-labeled goat anti-human IgA+G+M, washing and colorimetric detection of bound antibody using a Versamax spectrofluorometer (all steps were carried out at room temperature). The tested carbohydrate moieties included globo H, Lewis Y, Lewis X, sialyl-Thomson-nouveaux (sTn), clustered sTn, Thomson Friedenreich (TF), Tighe Le^(b)/Le^(Y) mucin, porcine submaxillary mucin (PSM), and sLe^(a) tetrasaccharide and sLe^(a)-HSA conjugate. To determine the fine specificity of the antibodies, glycan array analysis was done by the Consortium for Functional Glycomics Core H group. 5B1 and 7E3 antibodies were tested at 10 μg/mL using version 4.1 of the printed array consisting of 465 glycans in replicates of 6.

Immunoglobulin cDNA Cloning and Recombinant Antibody Expression

Variable region of human mAb heavy and light chain cDNA was recovered by RT-PCR from the individual hybridoma cell line and subcloned into IgG1 or IgM heavy chain or IgK or IgL light chain expression vector as described before. Sawada-Hirai et al., J. Immune Based Ther. Vaccines, 2:5 (2004). Ig heavy chain or light chain expression vector was double-digested with Not I and Sal I, and then both fragments were ligated to form a dual-gene expression vector. CHO cells in 6-well plates were transfected with the dual-gene expression vector using Lipofectamine 2000 (Invitrogen). After 24 hours, transfected cells were transferred to a 10-cm dish with selection medium [DMEM supplemented with 10% dialyzed FBS (Invitrogen), 50 μmol/L-methionine sulfoximine (MSX), GS supplement (Sigma-Aldrich), and penicillin/streptomycin (Omega Scientific)]. Two weeks later MSX-resistant transfectants were isolated and expanded. High anti-sLe^(a) antibody-producing clones were selected by measuring the antibody levels in supernatants in a sLe^(a)-specific ELISA assay and expanded for large-scale mAb production.

Human mAb Purification

Antibodies were purified using the Äkta Explorer (GE Healthcare) system running Unicorn 5.0 software. In brief, stable clones of 5B1 or 7E3 were grown in serum-free culture medium in a Wave bioreactor, and the harvested supernatant was clarified by centrifugation and filtration and stored refrigerated until used. Human IgG antibodies were purified on appropriate-sized protein A columns using 10 mmol/L PBS and 150 mmol/L NaCl running buffer. Human IgM antibodies were purified on a hydroxyapatite column, and IgM was eluted with a gradient of 500 mmol/L phosphate. The antibody concentrations were determined by OD₂₈₀ using an E^(1%) of 1.4 and 1.18 for IgG and IgM, respectively, to calculate the concentration. The purity of each preparation was evaluated by SDS-PAGE analysis (1-5 per lane) under reducing conditions, and the purity was more than 90% based on the sum of heavy and light chains.

Flow Cytometry

sLe^(a)-positive or -negative tumor cell lines (0.5×10⁶ cells per condition) were washed in PBS/2% FBS (PBSF). Test or control human mAb was then added (1-2 μg/mL in complete medium) and incubated on ice for 30 minutes. Gilewski et al., Clin Cancer Res, 6:1693-701 (2000); Gilewski et al. Proc. Natl. Acad. Sci. U.S.A., 98:3270-5 (2001). After washing in PB SF, the cells were incubated with Alexa-488 anti-human IgG-Fcγ or anti-human IgM-μ. (Invitrogen) for 30 minutes on ice. Cells were washed twice in PBSF and analyzed by flow cytometry using the Guava Personal Cell Analysis-96 (PCA-96) System (Millipore). Colo205-luc cells were incubated with 2 μg/mL of primary antibody, followed by staining with secondary antibodies from SouthernBiotech, and analyzed on a Becton Dickinson FACS Advantage IV instrument using FlowJo 7.2.4 software.

Affinity Determination

Affinity constants were determined using the principle of SPR with a Biacore 3000 (GE Healthcare). Biotin-labeled univalent sLe^(a) (Cat #02-044) or polyvalent sLe^(a)-PAA-biotin (Cat #01-044) were coupled to separate flow cells of an SPA biosensor chip according to the manufacturer's instructions. A flow cell blocked with HSA and culture medium containing free biotin was used as a reference cell. The binding kinetic parameters were determined from several known concentrations of antibody diluted in HBS-EP buffer (10 mmol/L HEPES, pH 7.4, 150 mmol/L NaCl, 3.4 mmol/L EDTA, 0.005% surfactant P20) using the sLe^(a)-PAA-biotin-coated flow cell. The curve-fitting software provided by the Biacore instrument was used to generate estimates of the association and dissociation rates from which affinities are calculated.

CDC Assay

sLe^(a) antigen-positive and -negative cell lines were used for a 90-minute cytotoxicity assay (Guava PCA-96 Cell-Toxicity kit; Millipore; Cat #4500-0200) using human complement (Quidel; Cat #A113) and purified human mAbs at various dilutions (0.1-25 μg/mL) or with positive control mAbs as previously described (Ragupathi et al. Clin Cancer Res 2003, 9:5214; Ragupathi et al. Int J Cancer 2000, 85:659; Dickler et al. Cancer Res 1999, 5:2773). In brief, 2.5×106 target cells were painted with carboxyfluorescein diacetate succinimyl ester (CSFE) to yield green/yellow fluorescent target cells. The painted cells (1×10⁵/50 μL sample) were incubated for 40 minutes with 100 μL of antibodies on ice. Next, 50 μL of human complement diluted 1:2 in complete medium (RPMI-1640, 10% FCS) or medium alone was added to triplicate samples and incubated for 90 minutes at 37° C. Thus, the final complement dilution in the assay was 1:8. Cells that were killed during this incubation time were labeled by adding the membrane impermeable dye 7-amino-actinomycin D (7-AAD), and samples were analyzed by dual-color immunofluorescence utilizing the Guava CellToxicity software module. Control samples that received NP40 were used to determine maximal killing and samples receiving complement alone served as baseline. The percentage of killed cells was determined by appropriate gating and calculated according to the following formula: % killed=[(% sample−% complement alone)/(% NP40−% complement alone)]×100.

Antibody-Dependent Cell-Mediated Cytotoxicity Assay

PBMC effector cells were isolated by Ficoll-Hypaque density centrifugation from blood samples obtained under an MSKCC IRB-approved protocol. The target cells were incubated at 5×10⁶ cells/mL in complete growth media with 15 μL of 0.1% calcein-AM solution (Sigma-Aldrich) for 30 minutes at 37° C., in the presence of 5% CO₂. The cells were washed twice with 15 mL of PBS-0.02% EDTA and resuspended in 1 mL complete growth medium. Fifty microliters (10,000 cells) of labeled target cells was plated into a 96-well plate in the presence or absence of antibodies at the concentrations described in FIG. 13A-13C, and incubated with 50 μL of freshly isolated peripheral blood mononuclear cells (effector cells, at 100:1 E/T ratio) accordingly. After 2 hours of incubation, the plate was centrifuged at 300×g for 10 minutes, and 75 μL of supernatant was transferred into a new flat-bottomed 96-well plate. The fluorescence in the supernatant was measured at 485-nm excitation and 535-nm emission in Fluoroskan Ascent (Thermo Scientific). Spontaneous release was determined from target cells in RPMI-1640 medium with 30% FBS without effector cells and maximum release was determined from target cells in RPMI-1640 medium with 30% FBS and 6% Triton X-100 without effector cells. Percent cytotoxicity was calculated as [(counts in sample−spontaneous release)/(maximum counts−spontaneous release)]×100.

mAb Internalization Assay

Internalization of 5B1 antibody was evaluated by measuring the cytotoxic activity of r5B1 and Hum-ZAP secondary conjugate (Advanced Targeting Systems) complex against sLe^(a) expressing BxPC3 cells, which were plated into a 96-well plate (2,000 cells/90 μL/well) and incubated overnight in duplicates. Various concentrations of 5B1 antibody were incubated with Hum-ZAP secondary conjugates at RT according to the manufacturer's instruction. Next, 10 μL/well of r5B1 and Hum-ZAP complex was added to the cells and incubated for 3 days. Twenty-five microliters of Thiazolyl Blue Tetrazolium Bromide (Sigma-Aldrich) solution (5 mg/mL in PBS) was added to each well and incubated at 37° C. After 2 hours of incubation, 100 μL/well of solubilization solution (20% SDS/50% N,N-dimethylformamide) was added to each well and incubated for another 16 hours at 37° C. The OD was measured at 570/690 nm, and values obtained with medium alone were used for plate background subtraction. Eight parallel cultures without antibody were used to normalize the sample values (sample/mean untreated×100).

Xenograft Transplantation Model

Female CB17 SCID mice (5-8 weeks old) were purchased from Taconic. For the Colo205 xenograft model, Colo205-luc cells (0.5×10⁶) in 0.1 mL complete growth media were injected via the tail vein on day 0 using a BD insulin syringe with 28G needle (Becton Dickinson & Co). For the first study, one hundred micrograms of mAb 5B1 was injected intraperitoneally on days 1, 7, 14, and 21 (experiment 1) or on days 1, 4, 7, 10, 14, and 21 (experiment 2). For the second study, 100 μg, 300 μg or 1 mg of mAb 5B1 was injected intraperitoneally on Day 4 after tumor cell injection, then twice a week for the first two weeks and once a week for the next 7 weeks. Mice were monitored for tumor development. For the DMS-79 xenograft model, DMS-79 cells (1×10⁶) were injected subcutaneously into Female CB17 SCID mice, and the mice began treatment on Day 19 after the tumor length reached 5 mm (˜20 mm²). The animals were then treated with human IgG or 5B1 antibodies given by intraperitoneal injection at 200 μg per dose, plus cRGD by intravenous injection to increase vascular permeability initially at 80 μg, then 5 days per week, 40 μg per dose until day 37.

All procedures were done under a protocol approved by the Memorial Sloan Kettering Cancer Center Institutional Animal Care and Use Committee. Kaplan-Meier survival curves were generated using GraphPad Prism 5.1 (GraphPad Software) and analyzed using the Mantel-Haenszel log-rank test.

Results Identification of Human Monoclonal Antibodies by ELISA and Generation of Recombinant Antibodies

Blood samples from 3 vaccinated patients were used for hybridoma generation efforts and many positive wells were detected in the antigen-specific ELISA assays (Table 3). Extensive screening was used to eliminate antibodies that showed inferior or nonspecific binding. Eight human antibody-expressing hybridoma cells (1 IgM and 7 IgG) with strong reactivity against sLe^(a) were initially selected, expanded, and subcloned for further characterization. Two antibodies (9H1 and 9H3) showed strong binding to sLe^(a)-HSA conjugates but not to sLe^(a)-PAA-coated plates. Three antibodies (5B1, 5H11, and 7E3) showed strong binding to monovalent and polyvalent sLe^(a) and sLe^(a)-HSA conjugates when measured by ELISA assays (Table 4).

TABLE 3 Binding of candidate hybridoma supernatants containing IgG or IgM monoclonal antibodies to sLeª-acetylphenylenediamine(APD)- human serum albumin(HSA) conjugate (sLeª-HuSA). OD (490 nm)* sLe^(a)- Supernatant Isotype HuSA HuSA PBS EF41-5B1 G 0.000 2.240 0.020 EF41-5H11 G 0.020 2.180 −0.010 EF41-6F7 G 0.010 0.480 −0.010 EF41-9H1 G 0.010 0.730 −0.020 EF41-9H3 G 0.010 1.100 −0.020 EF41-9A10 G 0.010 2.140 −0.010 EF41-10C1 G 0.000 0.040 −0.020 EF40-3C4 G 0.000 0.500 0.000 EF40-10H3 G 0.000 0.130 0.000 EF41-7E3 M −0.020 2.130 0.010 EF41-9A7 M 2.700 2.540 2.610 EF40-5B7 M 0.070 0.070 0.080 *isotype control blank subtracted. HuSA indicates human serum albumin control. PBS indicates phosphate buffered saline control.

TABLE 4 Binding of the select antibodies to sLeª presented as univalent (mono-) sLeª, multivalent (poly-) sLeª, or sLeª-HSA form. OD (490 nm) NAV + NAV + SLeA- Supernatant PBS NAV mono-sLeª poly-sLeª HSA* EF41-5B1(G) 0.050 0.050 0.900 2.280 1.740 EF41-5H11(G) 0.040 0.050 1.280 2.130 1.900 EF41-6F7(G) 0.050 0.050 0.050 0.080 0.100 EF41-9H1(G) 0.050 0.050 0.050 0.060 0.300 EF41-9H3 (G) 0.050 0.050 0.050 0.050 0.750 EF41-9A10 (G) 0.040 0.040 0.170 0.870 1.330 EF40-3C4 (G) 0.040 0.050 0.040 0.050 0.070 EF41-7E3 (M) 0.050 0.050 0.970 0.920 1.310 HuSA indicates human serum albumin control. PBS indicates phosphate buffered saline control. NAV indicates Neutral Avidin control.

The heavy and light chain variable regions from 4 selected antibodies were recovered by RT-PCR and cloned into our full-length IgG1 or IgM expression vectors. Molecular sequence analysis using IMGT/V-Quest (Brochet et al., Nucleic Acids Res., 36:W503-8 (2008)) revealed that the 3 selected IgG antibodies 5B1 (IgG/λ), 9H3 (IgG/λ), and 5H11 (IgG/λ) were derived from the same VH family and all used lambda light chains. These IgG1 antibodies showed different CDR sequences with 16, 5, or 3 mutations deviating from the germ line, respectively (FIGS. 1-6 ; Table 5). The IgM antibody (7E3) utilizes the kappa light chain and has 6 heavy chain mutations (FIGS. 7-8 ; Table 5). The increased mutations in 5B1 are indicative of affinity maturation. Recombinant antibodies were produced in CHO cell lines in a wave bioreactor system and purified using protein A or hydroxyapatite chromatography for IgG and IgM, respectively. The purified recombinant antibodies retained the properties of the original hybridoma-derived antibodies with respect to ELISA binding and specificity.

TABLE 5 cDNA Classification of selected human anti-sLe^(a) antibodies derived from vaccinated blood donors. VH VL Antibo. Muta. Muta. Clone from CDR from CDR ID VH germline DH (RF) JH length VL germline JL length 5B1 3-9*01 16 6-25*01 (1) 4*02 8, 8, 16 L1-47*01 4 JL1*01 8, 3, 12 9H3 3-9*01 5  2-8*01 (2) 4*02 8, 8, 16 L1-47*01 2 JL2*01 8, 3, 11 5H11 3-9*01 3 6-25*01(1)  4*02 8, 8, 16 L1-47*01 1 JL1*01 8, 3, 12 7E3 3-30*03  6 2-15*01 (2) 4*02 8, 8, 19 K3-15*01 3 JK2*01 6, 3, 10

Analysis of Tumor Cell Binding

Cell surface binding is crucial for cytotoxic activity and was therefore tested next. Flow cytometry showed strong binding of 5B1, 9H3, 5H11, and 7E3 recombinant antibodies to DMS-79 cells, a small-cell lung cancer suspension cell line (FIG. 11A). Binding of r5B1 and r7E3 was also confirmed on HT29 colon cancer cells (FIG. 11B), BxPC3 pancreatic cancer cells (FIG. 11C), SW626 ovarian cancer cells (FIG. 11D), and Colo205-luc colon cancer cells (FIG. 11F). These antibodies failed to bind to sLe^(a)-negative (SLE121-negative) SK-MEL28 melanoma cells (FIG. 11E) or EL4 mouse lymphoma cells (data not shown).

Affinity Measurements

The relative affinity/avidity of the binding to sLe^(a) was probed by SPR using a streptavidin-coated biosensor chip to capture biotinylated sLe^(a)-PPA. As shown in Table 6, r5B1 and r7E3 bind rapidly to sLe^(a)-PPA and show a significantly slower off-rate compared with 121 SLE, a commercially available murine IgM anti-sLe^(a) antibody that was used for comparison. The affinity of 5B1 was measured at 0.14 nmol/L, and the apparent affinity/avidity of 7E3 was approximately 4 times higher (Table 6). Determination of 9H3 affinity was hampered since 9H3 antibodies (native and recombinant) failed to bind to the sLe^(a)-PAA-coated biosensor chip.

TABLE 6 Determination of kinetic parameters of anti-sLe^(a) antibodies by SPR. Affinity, K_(d), K_(a), Association Dissociation mAb nmol/L mol/L 1/mol/L k_(a), 1/mol/L s) kd, 1/s Isotype r5B1 0.14 1.4 × 10⁻¹⁰ 7.0 × 10⁹ 1.1 × 10⁶ 1.6 × 10⁻⁴ IgG1/λ r7E3 0.04 3.6 × 10⁻¹¹  2.8 × 10¹⁰ 8.8 × 10⁵ 3.2 × 10⁻⁵ IgM/κ 121SLE 0.35 3.5 × 10⁻¹⁰ 2.8 × 10⁹ 2.7 × 10⁶ 9.4 × 10⁻⁴ m-IgM

Specificity Analysis

Preliminary assays to probe carbohydrate specificity showed that 5B1, 9H3, and 7E3 did not bind to the closely related sLe^(X), Le^(a), or Le^(Y) antigens or the gangliosides GD2, GD3, fucosyl-GM1, GM2, and GM3 as measured by ELISA or SPR. Additional analysis of 7E3, 5B1 and 121SLE binding to sLe^(a)-PAA-biotin or sLe^(a)-sp-biotin captured on a Biacore avidin chip showed that all three antibodies bound to the polyvalent form of sLe^(a), whereas 7E3 and 5B1 were found to bind the monovalent form. The binding of 5B1 to sLe^(a)-PAA was also inhibited by sLe^(a) tetrasaccharide in a dose-dependent manner in a Biacore concentration analysis series (data not shown). These results are consistent with previous observations that sera with high anti-sLe^(a) antibody titers were found to be specific for sLe^(a), that is, not reactive with gangliosides GM2, GD2, GD3, fucosyl GM1, or the neutral glycolipids globo H and Le^(y) by ELISA. Ragupathi et al., Cancer Immunol Immunother 58:1397-405 (2009). In a competition assay with 9 distinct related carbohydrate moieties in various presentations (e.g., as ceramide, or conjugated to BSA or HSA), only sLe^(a) tetrasaccharide and sLe^(a)-HSA conjugate were able to inhibit binding to sLe^(a)-HSA conjugate (Table 7).

TABLE 7 Binding to sLeA-PAA-HSA in the presence of various related glycoconjugates. r5B1 r9H3 r7E3 Antigens Exp 1 Exp 2 Exp 1 Exp 2 Exp 1 Exp 2 Sialyl Tn-HSA 1.866 1.981 1.882 1.970 2.218 2.259 GloboH-ceramide 1.866 1.852 1.906 1.821 2.098 2.201 sTn(c)-HSA (direct) 1.896 1.864 1.947 1.883 2.131 2.136 sTn-M2-HSA (mono) 1.937 1.857 1.843 1.826 2.040 2.066 LeX-gal-cer 1.893 1.863 1.791 1.810 2.173 2.175 dPSM 1.897 1.890 1.757 1.700 2.218 2.110 Tn-mono allyl M2-HSA 1.837 1.905 2.041 1.991 2.083 2.107 Tighe Leb/LeY mucin 1.808 1.837 1.951 1.964 2.106 2.065 LeX-PAA 1.830 1.873 2.053 2.036 2.099 2.108 LeY-ceramide 1.824 1.821 1.940 1.980 2.143 2.085 Lewis Y ceramide 1.833 1.844 1.941 1.874 2.090 2.111 Tn(c)-HSA 1.881 1.711 1.893 1.917 2.146 2.030 T-serine-BSA 1.809 1.830 2.128 2.089 2.137 2.039 TF(c) HSA 1.874 1.909 2.031 2.032 2.119 2.094 Tn LY-BSA 1.901 1.863 1.944 1.959 2.084 2.118 NPrGBMP-HSA 1.892 1.797 1.944 1.964 2.090 2.111 sLeA - HSA 1.329 1.298 1.373 1.266 1.542 1.621 sLeA tetrasaccharide 0.371 0.312 0.797 0.814 2.114 2.041 None 1.809 1.809 1.993 1.993 2.096 2.096 Blank 0.101 0.093 0.093 0.092 0.108 0.100

To examine the carbohydrate specificity in further detail, 5B1 and 7E3 antibodies were also tested by glycan array analysis done by the Consortium for Functional Glycomics Core H group. Both antibodies were tested at 10 μg/mL on printed arrays consisting of 465 glycans in 6 replicates. The results confirmed the high specificity of both antibodies with selective recognition of the sLe^(a) tetrasaccharide, Neu5Acα2-3Galβ1-3(Fucα1-4)G1cNAcβ and Neu5Gcα2-3Galβ1-3(Fucα1-4)G1cNAcβ and virtual absence of binding to closely related antigens that were present in the array, including sLe^(x), Le^(a), Lex, and Leg. The results are summarized in Table 8, which shows the top 5 of 465 glycan structures that were recognized by the respective antibodies.

TABLE 8 Analysis of carbohydrate specificity by glycan array screening. Chart Common Number Name Glycan Structure Average StDev % CV A. 5B1 237 sLeª Neu5Acα2-3GalB1-3(Fucα1-4)GlcNAcβ-Sp8 38,851 2,797 7 278 sLeª Neu5Gcα2-3GalB1-3(Fucα1-4)GlcNAcβ-Sp0 32,714 2,624 8 329 sLeªLeª Neu5Acα2-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1- 6,477 399 9 3(Fucα1-4)GlcNAcβ-Sp0 238 sLeªLe^(x) Neu5Acα2-3Galβ1-3(Fucα-4)GlcNAcβ1-3Galβ1- 1,344 131 10 4(Fucα1-3)GlcNAcβ-Sp0 349 Galβ1-4GlcNAcβ1-2Manα1-3(Manα1-6)Manβ1- 129 62 48 4GlcNAcβ1-4GlcNAcβ-Sp12 B. 7E3 237 sLeª Neu5Acα2-3Galβ1-3(Fucα1-4)GlcNAcβ-Sp8 40,920 4,676 11 329 sLeªLeª Neu5Acα2-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1- 40,210 2,095 5 3(Fucα1-4)GlcNAcβ-Sp0 238 sLeªLe^(x) Neu5Acα2-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1- 39,848 3,621 9 4(Fucα1-3)GlcNAcβ-Sp0 278 sLeª Neu5Gcα2-3Galβ1-3(Fucα1-4)GlcNAcβ-Sp0 36,707 2,733 7 349 Galβ1-4GlcNAcβ1-2Manα1-3(Manα1-6)Manβ1- 692 52 8 4GlcNAcβ1-4GlcNAcβ-Sp12

CDC Activity

To evaluate the functional activity of 5B1 and 7E3, we tested the cytotoxic activity with DMS-79 cells in the presence of human serum as a source of complement. Both antibodies showed in some assays close to 100% killing activity at 10 μg/mL, while a control antibody with different specificity (1B7, anti-GD2 IgG1 mAb) had no effect at the same concentrations (data not shown). The CDC activity is concentration dependent, and 7E3 was significantly more active than 5B1 in this assay (FIG. 12A), which is expected since IgM antibodies are known to be more effective in complement-mediated cytotoxicity assays. The EC50 (50% cytotoxicity) was 1.7 μg/mL for 5B1 and 0.1 μg/mL for 7E3, which translates to roughly 85-fold higher potency for 7E3 on a molar basis (FIG. 12B).

ADCC Activity

While 7E3 is significantly more potent in the CDC assay, IgG antibodies are known to have antibody-dependent cell-mediated cytotoxicity (ADCC) activity, which is thought to be important for tumor killing in vivo. High levels of cytotoxicity were measured using 5B1 antibody with human PBMC and DMS-79 target cells at various E:T ratios (FIG. 13A). Similar levels of cytotoxicity were observed at lower E:T ratios with primary NK cells (FIG. 13B). A dose-response experiment with PBMC from 2 donors measured at an E/T ratio of 100:1 showed similar efficacy, and more than 85% cytotoxicity was reached at concentrations of 0.5 μg/mL or more of 5B1 (FIG. 13C). The cytotoxicity mediated by 5B1 requires FcγRIII receptors since it can be blocked with 3G8 anti-CD16 antibodies. High levels of cytotoxicity were also measured using 5B1 antibody with human PBMC against Colo205-luc cells at an E:T ratio of 100:1. The ADCC activity achieved with 1 μg/mL of 5B1 antibodies was superior to the activity observed with antibodies to GM2, fucosyl-GM1, globo H, or polysialic acid. As expected, 7E3 and murine 121SLE (both are IgM) were inactive in this assay.

5B1 Internalization Assay

Antibody conjugates directed at antigen “closely related to” Lewis Y were previously shown to be rapidly internalized and very effective in animal models. Hellstrom et al., Cancer Res 50:2183-90 (1990); Trail et al., Science 261:212-5 (1993). To examine whether sLe^(a) is internalized, we incubated the pancreatic cell line, BxPC3 with 5B1, and then added Hum-ZAP, an anti-human IgG conjugated to the ribosome-inactivation protein saporin. Kohls et al., Biotechniques 28:162-5 (2000). Cells that internalize the saporin-containing complex die, while noninternalized saporin leaves the cells unharmed. As shown in FIG. 14 , BxPC3 cells are effectively killed in the presence of increasing doses of 5B1 while the presence of an isotype-matched IgG1 antibody directed against GD2, which is not expressed on these cells, does not kill the cells.

Activity in Xenograft Animal Model for Metastasis

To evaluate the activity of 5B1 in vivo, the antibodies were tested in two xenograft models using either Colo205-luc tumor cells or DMS-79 tumor cells in SCID mice. For the xenograft model using Colo205-luc tumor cells, five mice per group were injected with 0.5×10⁶ cells into the tail vein on day 0, and successful injection of the cells was verified by imaging the animals using the IVIS 200 in vivo imaging system (Caliper Life Sciences). One day later, animals were treated with 5B1 antibodies given intraperitoneal or PBS mock injection. In experiment 1, 100 μg of 5B1 was given on days 1, 7, 14, and 21 (400 μs total dose), and in experiment 2 the animals received 100 μg 5B1 on days 1, 4, 7, 10, 14, and 21 (600 μg total dose). The average median survival of untreated animals was 102 days in the 2 experiments, and all untreated animals died within 155 days (FIG. 15 ). Treatment of animals improved survival significantly: the median survival was doubled to 207 days in the group that received 4 doses of 5B1 and 2 of 5 animals survived until termination of the experiment after 301 days (log-rank test, P=0.0499; HR=3.46). The proportion of survivors further increased to 3 of 5 mice when 6 doses were administered (log-rank test, P=0.0064; HR=6.375). The second experiment was terminated after 308 days, and the surviving animals failed to reveal Colo205-luc tumors at the highest sensitivity of the imaging system (data not shown).

In a second study, mice similarly injected with Colo205-luc tumor cells as described above, were treated with increasing doses of 5B1 or 7E3 antibodies (100 μg, 300 μg or 1 mg). All animals initially received interperitoneal or PBS mock injection (control) of the 5B1 or 7E3 antibody on Day 4 after tumor cell injection, then twice a week for the first two weeks and once a week for the next 7 weeks. The delayed treatment with various doses of 5B1 showed a dose dependent protection up to complete cure in SCID mice engrafted with Colo205-luc tumor cells (FIGS. 16 and 17 ). Treatment with 7E3 antibodies did not show higher protection despite increased apparent affinity (data not shown).

In a xenograft model using DMS-79 cells, five mice per group were injected subcutaneously with 1×10⁶ cells on day 0, and began treatment on day 19 after the tumor length reached 5 mm (˜20 mm²). The animals were then treated with human IgG or 5B1 antibodies given by intraperitoneal injection at 200 μg per dose, plus cRGD by intravenous injection initially at 80 μg, then 5 days per week, 40 μg per dose until day 37. The growth of established DMS-79 tumors was suppressed or regressed in animals treated with 5B1 or a combination of 5B1 plus cRGD (FIGS. 18A and 18B). Treatment of animals with 5B1 on the day of engraftment with DMS-79 cells in a subcutaneous model completely prevented tumor growth (data not shown).

The above data demonstrates a significant ability to suppress or regress established tumors and provide a survival benefit using 5B1 antibody treatment.

Example II Immuno-PET Detection and Diagnosis of Pancreatic Cancer and Other sLe^(a) Positive Adenocarcinomas Using Radiolabeled Monoclonal Antibody 5B1

Adenocarcinomas are a leading cause of death from cancer. Detection of pancreatic cancer remains especially difficult with diagnosis often made at a late stage. Approaches for earlier detection of primary and metastatic pancreatic cancers could have significant clinical impact. In clinical practice, elevated levels of sLe^(a) antigen are monitored to identify suspected occult malignancy in patients with pancreatic cancer. As described herein, the potential of a novel immunoPET imaging probe targeting sLe^(a) in preclinical models of pancreatic cancer and other sLe^(a) positive adenocarcinomas was investigated. The human anti-sLe^(a) monoclonal antibody 5B1 showed positive staining on human adenocarcinomas known to be sLe^(a) positive but not on sLe^(a) negative malignancies or most normal tissues. ⁸⁹Zr-radiolabeled 5B1 (⁸⁹Zr-5B1) displayed high labeling (>80%) and purification yields (>95%). Imaging with ⁸⁹Zr-5B1 was investigated in subcutaneous, orthotopic and metastatic pancreatic cancer xenografts in female SCID mice. Acquired PET images and biodistribution studies demonstrated exceptional specificity and localization of ⁸⁹Zr-5B1 for the sLe^(a) overexpressing BxPC3 xenografts with minimal non-specific binding to healthy tissues. Further analysis in colon and small cell lung cancer subcutaneous xenograft models resulted in excellent tumor delineation by ⁸⁹Zr-5B1 as well. Accordingly, these results show that ⁸⁹Zr-5B1 can be used as a molecular probe for early detection of sLe^(a) expressing malignancies in the clinic.

Cell Lines and Tissue Culture

All tissue culture manipulations were performed following sterile techniques. The small cell lung cancer DMS79 and BxPC3 pancreas cancer cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Colo205-luc colorectal cancer cells (Bioware Ultra) were purchased from Caliper Life Sciences (CLS, Hopkinton, MA). All cells were grown according to the recommendations of ATCC and CLS under 37° C. with 5% CO₂ humidified atmosphere.

In Vitro Evaluation of sLe^(a) Expression Levels Through FACS

Flow cytometry with the indicated cultured cancer cell lines was performed as described herein in Example I. In brief, single cell suspensions of 1×10⁶ culture tumor cells per tube were washed in PBS with 3% fetal bovine serum (FBS). Human monoclonal antibodies r5B1 (IgG against sLe^(a)) was then added at 20 ug/ml per tube, and incubated on ice for 30 min. After wash in PBS with 3% FBS, 20 μl of 1:25 diluted goat anti-human IgG labeled with fluorescein-isothiocyanate (FITC, Southern Biotechnology, Birmingham, AL) was added, and the mixture incubated for another 30 minutes on ice. After a final wash, the positive population and median fluorescence intensity of stained cells were differentiated using FACS Scan (Becton & Dickinson, San Jose, CA). Cells stained only with goat antihuman IgG labeled with fluorescein-isothiocyanate were used to set the FACScan result at 1% as background for comparison to percent positive cells stained with primary mAb.

Preparation of ⁸⁹Zr-Labeled Antibodies

Recombinant 5B1 antibodies was prepared and purified as described herein. The 5B1 antibodies and a non-specific human IgG were functionalized with p-isothiocyanatobenzyl-desferrioxamine (DFO-Bz-NCS, Macrocyclics, Inc., Dallas, TX) with a 1:4 mAb:DFO-Bz-NCS ratio. For example, to 300 μL of 5B1 (1.23 mg in PBS, pH˜9), a volume of 7.2 μL DFO-Bz-NCS (4.25 mM in DMSO) was added. The reaction was incubated at 37° C. for 1-1.5 h. The functionalized antibodies were purified via either PD10 desalting column (GE Healthcare) or a 10 kDa centrifugal filter (Amicon).

Zr-89 was produced through proton beam bombardment of yttrium foil and isolated in high purity as Zr-89 oxalate at MSKCC according to previously established procedure. Holland et al., Nuclear Medicine and Biology 36:729-39 (2009). Labeling of the antibodies proceeded via methods as described by Holland et al., Journal of Nuclear Medicine official publication, Society of Nuclear Medicine 51:1293-300 (2010). In general, Zr-89 oxalate was neutralized to pH 7.0-7.2 with 1 M Na₂CO₃. The DFO-antibodies were then added. The reaction was incubated at room temperature for 1-2 hours. Subsequent purification was conducted using either a PD10 desalting column with 0.9% saline.

In Vitro Experiments

⁸⁹Zr-5B1 was investigated for stability in vitro in 0.9% saline and in 1% bovine serum albumin for 5 days at 37° C. Changes in radiochemical purity were monitored at t=0-5 days via radio iTLC with 50 mM DTPA as mobile phase. In vitro immunoreactivity assays were performed according to the protocol described by Lindmo et al., Journal of Immunological Methods 72:77-89 (1984), to demonstrate the integrity of the Zr-89 radiolabeled antibodies.

Animal Models

All animal studies were conducted in accordance with the guidelines set by the Institutional Animal Care and Use Committee. Female CB17SC-F SCID mice (Jackson Laboratories, 6-8 weeks, 20-22 g) or nude athymic (nu/nu) mice were induced with tumors on hind legs. All cell lines were inoculated subcutaneously in 200 μL of 1:1 media:Matrigel (BD Biosciences) solution and grown to a maximum tumor volume of 250 mm³ before use.

Biodistribution Studies

Biodistribution studies were performed on several cohorts of mice bearing separate Colo205-luc colorectal, BxPC3 pancreas and DMS79 small cell lung xenografts (n=3-5). Zr-89 mAbs (10-20 μCi, 1-2 μg) in 100 μL 0.9% saline were administered intravenously in the lateral vein. Additional unlabeled mAb (10-50 μg) was co-injected along with the tracer. A blocking study with 250 μg excess of unlabeled mAb was performed to address specificity of the antibody to sLe^(a) in a cohort of mice. After each time point (t=24, 48, 120 h p.i.), the mice were euthanized by asphyxiation with CO₂. Blood was collected immediately via cardiac puncture while the tumor along with chosen organs was harvested. The wet weight of each tissue was measured, and the radioactivity bound to each organ was counted using a Wizard² 2480 gamma counter (Perkin Elmer). The percentage of tracer uptake expressed as % injected dose per gram (% ID/g) was calculated as the activity bound to the tissue per organ weight per actual injected dose decay-corrected to the time of counting.

Small Animal Immuno-PET

Imaging experiments were accomplished with a microPET Focus 120 or R4 scanner (Concorde Microsystems). Mice (n=3-5) were administered Zr-89 labeled antibodies (200-300 μCi, 15-25 μg) in 100-200 μL 0.9% saline formulations via lateral tail vein injections. PET whole body acquisitions were recorded on mice at 24-96 h p.i. while anesthetized with 1.5-2.0% isofluorane (Baxter Healthcare) in oxygen. The images were analyzed using ASIPro VM™ software (Concorde Microsystems). Regions-of-interest (ROI) were drawn and plotted vs. time.

Immunohistochemistry

Biotinylated 5B1 was prepared by incubating 20× molar excess Sulfo-NHS-LC-biotin (Thermo Scientific/Pierce cat #21327) for 30 minutes at room temperature. Free biotin was removed with Zebra™ desalt spin columns (Thermo Scientific/Pierce, cat #89889) according to the manufacturer's instructions. The antibodies were buffer exchanged to PBS containing 0.01% sodium azide at a concentration of 1.1 mg/ml. The binding on DMS79 cells was confirmed by FACS and was comparable to the parent 5B1 antibody.

Preliminary immunohistochemistry staining conditions were determined using Colo205 cells as positive control and SK-MEL28 cells as negative control. Cell pellets were prepared, formalin fixed and paraffin embedded. The slides were incubated with biotinylated 5B1 diluted in 10% (v/v) normal human serum in PBS (Jackson ImmunoResearch Labs; cat #009-000-121). The staining was performed by Ventana automation (Discovery XT platform-Ventana Medical Systems, Inc, Tucson, AZ) with standard streptavidin-biotin immunoperoxidase method and DAB detection system as a staining method. Antigen recovery was conducted using heat and Ventana's CC1 conditioning solution. CA 19.9 mouse monoclonal (clone 116-NS-19-9) from Signet (Covance) gave comparable results in the pilot study. Colo205 cells are strongly positive with biotinylated 5B1 used at 10 μg/ml while SKMEL28 cells were completely negative. Histo-Array™ tissue microarrays were purchased from Imgenex (San Diego, CA). The following slides containing tumor biopsy cores as well as some normal tissue cores were used: IMH-327 (Common Cancers, 59 samples), IMH-359 (colorectal: cancer-metastasis-normal; 59 samples), and IMH-324 (Metastatic cancer to ovary). Pancreatic tumor tissue cores were present on IMH-327.

sLe^(a) Serum Concentration In Vivo

Mice bearing xenografts of Colo205, BxPC3 and DMS79 were exsanguinated for sLe^(a) antigen assays. A group of mice with no tumor served as a control. The sLe^(a) levels in the sera of mice were measured using the ST AIA-PACK CA19.9 kit (Cat #025271, TOSOH Bioscience Inc, South San Francisco, CA). The principle of the assay is based on the two site immunoenzyme-metric assay. The analysis was performed as described in the manufacturer's instruction manual. The optical density of immunoassay plates were measured by TOSOH AIA2000 Automated immunoassay analyzer (TOSOH Bioscience, Inc, San Francisco, CA).

Statistical Analysis

Data values were expressed as the mean±SD unless otherwise stated. Statistical analysis was performed using GraphPad Prism version 5.03 software using one-way ANOVA followed by Dunnett test. A P value of <0.05 is considered statistically significant.

Results

The binding specificity of 5B1 was probed by staining selected malignant and normal tissue microarrays. 5B1 reactivity was restricted to malignancies and occasional normal tissues previously known to overexpress sLe^(a) (FIGS. 19A-19F; Table 9). Most normal tissues were completely negative (Table 9). In contrast, strong positive staining was found in 21/34 colon adenocarcinomas (62%), 33/57 adenocarcinoma metastases to the ovary (58%), and 7/9 pancreatic ductal cancers (66%) at various stages (Table 10). As shown in FIGS. 19A-19F, typical reactivity was diffuse cytoplasmic staining with some tumor cells clearly showing distinct staining of the cell membrane. In addition, some signet ring ovarian cancers, and some cancers of the lung and breast were also found to be strongly positive. In contrast, only 4/43 prostate cancer samples and 0/51 GIST cases were positive (data not shown).

TABLE 9 Survey of 5B1 binding to normal tissues. Normal Tissue Stain Brain negative Breast positive Colon positive Kidney negative Liver negative Lung negative Lymph node negative Muscle negative Pancreas positive Placenta negative Skin negative Spleen negative Stomach negative

TABLE 10 Staining of Pancreatic Ductal Adenocarcinomas with 5B1. IHC 5B1 Stage Age Sex Histology neg II 71 M moderately differentiated pos++ III 68 M moderately differentiated neg III 64 F moderately differentiated pos++ III 46 M moderately differentiated pos++ III 54 M moderately differentiated pos++ III 40 M moderately differentiated pos+/− IVA 66 M moderately differentiated pos++ IVA 45 M moderately differentiated poor tissue IVA 64 F moderately differentiated pos++ IVA 69 M poorly differentiated

The high specificity of 5B1 immunostaining for cancer tissues expressing sLe^(a) was the basis for using this mAb as a PET probe. Modification of 5B1 with the benzyl-isothiocyanate analog of desferrioxamine (DFO-Bz-NCS) was made at a ratio of 4:1 (chelate:mAb) with subsequent purification via centrifugal filtration using saline as the washing buffer. Facile radiolabeling with Zr-89 proceeded at room temperature after pH adjustment to 7.0-7.2. A narrower pH range closer to neutral is necessary to achieve optimum radiolabeling yields of >80%. Free, unbound Zr-89 was removed via PD10 desalting column. Concentration of the product was made using a centrifugal filter (MWCO: 10 kDa). A relatively high specific activity of 12.1±1.1 mCi/mg was established. Radiochemical purities of more than 95% were ensured prior to use. Immunoreactivity assays displayed retention of activity for sLe^(a) (72.4±1.1%, n=3). Stability in bovine serum albumin at 37° C. was maintained at >95% over 5 days (data not shown). In saline, de-metalation was observed as early as 24 h (>85% complexed) with about >75% radiometal bound after 120 h at 37° C.

Small animal PET imaging and biodistribution studies were conducted using female SCID mice subcutaneously implanted with BxPC3 pancreas cancer xenografts on the left hind leg. Acquired PET images confirmed substantial delineation of the tumor-associated sLe^(a) by ⁸⁹Zr-5B1. From the maximum intensity projections (MIP) in FIG. 20 , the BxPC3 xenografts (n=3) showed exceptional accretion of the radiotracer administered intravenously. Regions-of-interest (ROI) drawn on the tumor from the PET images displayed an uptake of 5.0±0.4% ID/g (2 h), 16.2±2.5% ID/g (24 h), 23.8±4.7% ID/g (48 h), 36.8±6.1% ID/g (96 h) and 49.5±7.7% ID/g (120 h). Blood pool and normal tissue binding activity appeared to clear after 24 h p.i. Results from the biodistribution experiments are consistent with the PET data. High tumor localization of ⁸⁹Zr-5B1 at 24 h (84.7±12.3% ID/g, n=4) was observed; increased uptake was exhibited further at 120 h p.i. (114.1±23.1% ID/g, n=4) (FIG. 21 ). The tumor uptake exceeds 100% due to the small weight (62.4±0.03 mg). The % ID at 24 h p.i. was found to be ten-fold higher than that of the non-specific IgG at similar time points (FIG. 21 Inset). Competitive inhibition with 250 μg of non-radiolabeled 5B1 at 24 h p.i. blocked the tracer accumulation defining the specificity of uptake. Minimal binding of the ⁸⁹Zr-5B1 to normal pancreas and the rest of the harvested normal tissues was observed, providing a high tumor-to-tissue contrast at all time points.

Following the above results, ⁸⁹Zr-5B1 was assayed in an orthotopic BxPC3 pancreas tumor model. Orthotopic models are clinically relevant and offer an clinically accepted test of the efficacy of the PET probe. After inoculation in the pancreas, the tumor growth was monitored weekly via bioluminescent optical imaging. PET imaging experiments were conducted once the tumors are palpable. A comparison of probe tumor delineation properties were made between FDG-PET and ⁸⁹Zr-5B1 (FIGS. 25A-25B). Computed tomography (CT) in tandem with PET afforded an enhanced visualization of the anatomic region of interest.

To evaluate ⁸⁹Zr-5B1 as a PET probe in other sLe^(a) expressing adenocarcinomas, ⁸⁹Zr-5B1 was assayed in lung and colon cancer models. Small animal experiments were conducted using DMS79 small cell lung cancer cells and Colo205-luc colon cancer cells injected subcutaneously on the right hind leg of female SCID mice. PET MIP images were acquired after 24-120 h p.i. of 200-300 μCi (16-25 μg) injected intravenously. Heterogeneous DMS79 tumor uptake was demonstrated with 38.15±2.12% ID/g as early as 24 h p.i with excellent signal against background (FIG. 22A). An increase in tracer tumor accumulation resulted after 48 h p.i. (44.60±6.47% ID/g) with retention at 120 h p.i. (41.97±12.23% ID/g). Non-specific bound ⁸⁹Zr-5B1 cleared rapidly from normal tissues with minimal to no background uptake at 48 h p.i. In addition, tumor delineation was observed in the Colo205-luc xenografts as shown in FIG. 22B at 24-120 h p.i. The ROIs displayed tumor accumulation with 10.5±0.76, 23.5±2.7, 24.8±4.0, 18.4±4.7, 16.5±2.3% ID/g at 2, 24, 48, 96 and 120 h respectively. An observable increase in liver accumulation resulted over time with consequent decrease in tumor uptake as shown in the regions-of-interest drawn from the PET images (FIG. 22C). Data generated from the biodistribution studies correlate well with the observed PET results (data not shown).

The sLe^(a) level in mouse serum as tumors progressed was quantified. Exsanguination of SCID mice bearing Colo205, DMS79 and BxPC3 xenografts with a non-tumor bearing group serving as control was performed. sLe^(a) values showed high levels of sLe^(a) in mice challenged with Colo205 in comparison to the pancreatic BxPC3 and DSM79 implanted mice (Table 11).

TABLE 11 sLeª serum values from mice bearing colorectal (Colo205), pancreas (BxPC3) and small cell lung (DMS79) tumor xenografts compared to control. Tumor type Animal # Tumor volume, mm³ sLeª, U/ml Colo205-luc M1 269.5 3227 M2 257.3 2957 M3 281.3 1318 BxPC3 M1 232.38 N.D. M2 320.00 N.D. M3 220.50 N.D. DMS79 M1 288.0 N.D. M2 245.0 N.D. M3 232.4 N.D. Control M1 — 3 M2 — 3 M3 — 3 N.D. = Not detected.

These results demonstrate that a radiolabeled anti-sLe^(a) antibody (⁸⁹Zr-5B1) is specific for the detection and diagnosis of pancreatic adenocarcinoma and other sLe^(a) positive adenocarcinomas. ⁸⁹Zr-5B1 was produced with excellent yields and purity, along with high specific activity and retained immunoreactivity. Evaluation of ⁸⁹Zr-5B1 in subcutaneous, orthotopic and metastatic pancreas tumor models afforded excellent tumor delineation and diagnosis. Pre-clinical evaluation of this radiotracer in colon and small cell lung tumor-bearing small animals demonstrated the universal utility of this tracer for malignancies expressing sLe^(a).

Example III Anti-sLe^(a) Diabodies Bind to Various Cancer Cell Lines

Two diabodies were generated using the VH and VL domains of 5B1 and 7E3 clonal isolates described herein, designated 5B1CysDb and 7E3CysDb, respectively (FIGS. 9 and 10 ). Both diabodies contained a five amino acid linker region between the VL and VH domains. A poly histidine tag on the C-terminal, which was utilized for purification and detection, was also included for both diabodies.

The binding of 5B1CysDb and 7E3CysDb to three cancer cell lines: (1) DMS-79 cells, a small-cell lung cancer suspension cell line; (2) Capan-2 cells, pancreatic adenocarcinoma cells; and (3) BxPC3 cells, pancreatic cancer cells, was assayed by incubating 0.25 million cells in 0.2 ml with 10 μg/ml 5B1CysDb or 7E3CysDb, respectively. The cell and diabody combinations were incubated for 40 minutes on ice in PBS/2% FBS.

After washing, the cells were incubated for 40 minutes with 0.2 ml ALEXA-488-labeled anti-His antibody diluted 1:1000 (Life Technology, Cat #A21215). Following a second wash, the cells were analyzed with a Guava Flow Cytometer. Both 5B1CysDb and 7E3CysDb demonstrated significant binding to DMS-79, Capan-2 and BxPC3 cells (Table 12).

TABLE 12 Binding of 5B1CysDb and 7E3CysDb to Cell Lines 5B1CysDb 7E3CysDb Cell line Percent (+) MFI Percent (+) MFI DMS-79 98.1 113.0 93.8 124.6 Capan-2 63.8 98.5 65.9 235.3 BxPC3 51.3 39.9 50.2 49.7 MFI—mean fluorescent intensity

Example IV Administration of 5B1 and Taxol Inhibits Tumor Growth

The anti-tumor activity of co-administrating an anti-sLe^(a) antibody (5B1) and the chemotherapeutic agent Taxol (Paclitaxel) was assessed in xenograft models of pancreatic cancer and small cell lung cancer. As described previously herein, 1 million BxPc3 cells (pancreatic tumor cells) or 5 million DMS-79 cells (small cell lung cancer cells) were injected into the hind flank of 6 weeks old female CB17 SCID mice (Day 0; N=5). DMS79 tumors were allowed to grow for 21 days until the average tumor size was 193±64 mm3. Human IgG or 5B1 (0.5 or 1 mg) was given ip twice a week (strating on Day 21), and Taxol (0.2 mg/dose) was administered iv on days 23,30, 37 and 44. In the DMS-79 xenograft model, co-administration of 5B1 antibody and Taxol significantly limited tumor growth and resulted in tumor regression in comparison to control human IgG or 5B1 antibody and Taxol administered individually (FIG. 23 ).

In the BxPc3 xenograft model, tumors were grown for 14 days, at which they reached an average of 126±30 mm3. Taxol was administered iv on days 14, 21, 28 and 34 (weekly) and 5B1 was given twice per week starting on day 14. Co-administration of 5B1 antibody and Taxol significantly limited tumor growth in comparison to controls or 5B1 antibody and Taxol administered individually (FIG. 24 ). These results demonstrate a synergistic effect of an anti-sLe^(a) antibody and a chemotherapeutic agent in preventing tumor growth and/or reducing tumor size for pancreatic and small cell lung cancers.

Throughout this application various publications have been referenced. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains. Although the invention has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the spirit of the invention. 

1.-24. (canceled)
 25. A method of cancer treatment, the method comprising a step of: co-administering to a human subject having cancer first and second therapeutic agents, wherein the first therapeutic agent is or comprises a human antibody or antigen binding fragment thereof that binds to sialyl-Lewis^(a), which antibody or antigen binding fragment thereof comprises: a variable heavy chain domain having CDRs that include: (i) CDR1 represented by amino acid residues 55-62 of SEQ ID NO: 2; (ii) CDR2 represented by amino acid residues 70-77 of SEQ ID NO: 2; and (iii) CDR3 represented by amino acid residues 116-131 of SEQ ID NO: 2; and a variable light chain domain having CDRs that include (i) CDR1 represented by amino acid residues 45-52 of SEQ ID NO: 4; (ii) CDR2 represented by amino acid residues 70-72 of SEQ ID NO: 4; and (iii) CDR3 represented by amino acid residues 109-120 of SEQ ID NO: 4; and wherein the second therapeutic agent is or comprises a chemotherapeutic agent.
 26. The method of claim 25, wherein the first and second therapeutic agents are administered concurrently.
 27. The method of claim 25, wherein the first and second therapeutic agents are administered successively.
 28. The method of claim 27, wherein the step of co-administering comprises administering the first therapeutic agent to a subject who is receiving the second therapeutic agent.
 29. The method of claim 27, wherein the step of co-administering comprises administering the second therapeutic agent to a subject who is receiving the first therapeutic agent.
 30. The method of claim 25, wherein the first therapeutic agent is administered intravenously.
 31. The method of claim 25, wherein the cancer is a sialyl-Lewis^(a)-positive cancer.
 32. The method of claim 31, wherein the cancer is selected from the group consisting of a tumor of the gastrointestinal tract, colon cancer, colorectal adenocarcinoma, metastatic colon cancer, colorectal cancer, pancreatic cancer, pancreatic adenocarcinoma, small cell carcinoma of the lung, bladder adenocarcinoma, signet ring ovarian cancer, ovarian cancer, metastatic carcinoma, adenocarcinoma of the stomach, adenocarcinoma of the esophagus, adenocarcinoma of the throat, adenocarcinoma of the urogenital tract, and adenocarcinoma of the breast.
 33. The method of claim 25, wherein the subject is suffering from pancreatic cancer.
 34. The method of claim 25, wherein the subject is suffering from colorectal cancer.
 35. The method of claim 25, wherein the subject is suffering from small cell lung cancer.
 36. The method of claim 25, wherein the subject is suffering from or susceptible to cancer metastasis.
 37. The method of claim 25, wherein the variable heavy chain domain has an amino acid sequence represented by amino acid residues 20-142 of SEQ ID NO: 2 or a derivative thereof comprising less than 10 amino acid substitutions relative to the amino acid sequence represented by amino acid residues 20-142 of SEQ ID NO:
 2. 38. The method of claim 25, wherein the variable light chain domain has an amino acid sequence represented by amino acid residues 20-130 of SEQ ID NO: 4 or a derivative thereof comprising less than 10 amino acid substitutions relative to the amino acid sequence represented by amino acid residues 20-130 of SEQ ID NO:
 4. 39. The method of claim 25, wherein the first therapeutic agent is or comprises the antibody that binds to sialyl-Lewis^(a).
 40. The method of claim 39, wherein the antibody is a monoclonal antibody.
 41. The method of claim 39, wherein the antibody is an IgG isotype.
 42. The method of claim 41, wherein the IgG isotype is IgG1.
 43. The method of claim 25, wherein the first therapeutic agent is or comprises the antigen binding fragment thereof that binds to sialyl-Lewis^(a).
 44. The method of claim 43, wherein the antigen binding fragment is selected from the group consisting of a Fab, a Fab′, a F(ab′)₂, a scFv, a diabody, a triabody, a minibody and a single-domain antibody (sdAB).
 45. The method of claim 25, wherein the chemotherapeutic agent is or comprises a taxane.
 46. The method of claim 45, wherein the taxane is or comprises paclitaxel.
 47. The method of claim 25, wherein the human subject has a circulating level of sLe^(a) antigen that is indicative of the cancer.
 48. The method of claim 31, wherein the variable heavy chain domain has an amino acid sequence represented by amino acid residues 20-142 of SEQ ID NO: 2 or a derivative thereof comprising less than 10 amino acid substitutions relative to the amino acid sequence represented by amino acid residues 20-142 of SEQ ID NO:
 2. 49. The method of claim 31, wherein the variable light chain domain has an amino acid sequence represented by amino acid residues 20-130 of SEQ ID NO: 4 or a derivative thereof comprising less than 10 amino acid substitutions relative to the amino acid sequence represented by amino acid residues 20-130 of SEQ ID NO:
 4. 50. The method of claim 31, wherein the first therapeutic agent is or comprises the antibody that binds to sialyl-Lewis^(a).
 51. The method of claim 31, wherein the first therapeutic agent is or comprises the antigen binding fragment thereof that binds to sialyl-Lewis^(a).
 52. The method of claim 31, wherein the chemotherapeutic agent is or comprises a taxane.
 53. The method of claim 52, wherein the taxane is or comprises paclitaxel. 